COLUMBIA  LIBRARIES  OFFSITE 

HEALTH  SCIENCES  STANDARD 


HX64083195 
QM551  .Sch1  1897  A  course  of  practica 


THE 

LIBRARY 

OF  THE 

ASSOCIATION 

r^JSHic^.  OF  THE 

I  ALUMNI  i 

'nil  11  \\> 

OF  THE 

COLLEGE 

OF 

PHYSICIANS  AND 
SURGEONS 

IN  THE 

CITY  OF  NEW  YORK 


SCHOOL  OF  MEDICINE  OF  COLUMBIA  UNIVERSITY 


ALOMNI  ASSOCIAl.ui>, 

COLLEGE  OF  PHYSIClAi'^3  /\.,L)bu.\,     -' 
COLUMBIA  UMVE.<Sii  Y 
NEW  YORK 


PRACTICAL    HISTOLOGY 


Digitized  by  tine  Internet  Arciiive 

in  2010  witii  funding  from 
Columbia  University  Libraries 


http://www.archive.org/details/courseofpracticaOOshar 


frogcttg  0t  t^ie 
^s^otmtion  of  t\t  %[mn 

ire^iuB  13S  i.  OL.  |"tb 

OP 


PRACTICAL    HISTO 


BY 


EDWARD  ALBERT  SCHAFER,  LL.D.,  F.R.S. 

JODEELL     PROFESSOR     OF     PHYSIOLOGY     IN 
UXn-ERSITY    COLLEGE,    LONDON 


SECOND    EDITION 


PHILADELPHIA 

LEA    BEOTHEES    &    CO. 
1897 


UBRARV  OF  THE 

ALUmNi  ASSOCIATIUN, 

rOlLEGEOF  PHV8ICIA:  -.  .--abi^UN. 

COLUMBIA  UNlVERSllY 
NEW  YORK 


PEEFACE 


The    DUrDOSe    of    this    work    is    tn    aflfm-rl    frx    filler.    ^-^^^^^A 


Corrigenda 

Page    15,  line  8  from  top,  for  mammalian  read  human 

87,     „    14-15  from  top,  /o?-  Salamander  read  Salaniandra 
„    255,     „    1,  after  eosin  insert  a  comma 
„    266,     „    17,  for  methyl-blue  read  methylene-blue 


Schafer's  Practical  Histology. 


±xjL    a,    jjaiuiuuiar  liuB  oi    researca.       ivioreover,    sncli   anT 
account  would  have  been  unnecessary,  since  (to  the  great 
convenience  of  investigators)  it  is  already  furnished  by 
Dr.  A .  Bolles  Lee  in  his  book  '  The  Microtomist's  Vade 
Mecum.' 

In  an  Introductory  Chapter  an  account  is  given  of  the 
several  parts  of  the  microscope,  and  the  purpose  for  which 


ALUmNTASSOClATluN, 

C01LEGE0F?HYtilCIAr:6  ^  .^ -^.;',^'-""' 
COLUWIBIA  UNIVERSIIV 
NEW  YORK 


PEEFACE 


The  purpose  of  this  work  is  to  afford  to  those  engaged 
in  the  practical  study  of  Histology  plain  and  intelligible 
directions  for  the  suitable  pi-eparation  of  the  animal 
tissues,  with  the  object  either  of  immediate  study  or  of 
their  preservation  as  specimens  for  future  reference.  The 
aim  throughout  has  been  to  assist  the  student  to  carry  on 
histological  work  independently  of  the  constant  presence 
of  a  teacher.  Care  has  therefore  been  taken  to  select 
methods  upon  which,  in  the  author's  experience,  complete 
reliance  can  be  placed- — ^ provided,  of  course,  that  the 
directions  given  are  faithfully  followed  ;  and  no  attempt 
has  been  made  to  give  anything  approaching  a  complete 
account  of  modern  methods,  the  multiplicity  of  which  is 
bewildering,  and  many  of  which  have  been  only  devised 
for  a  particular  line  of  research.  Moreover,  such  an 
account  would  have  been  unnecessary,  since  (to  the  great 
convenience  of  investigators)  it  is  already  furnished  by 
Dr.  A.  Bolles  Lee  in  his  book  'The  Microtomist's  Vade 
Mecum.' 

In  an  Introductory  Chaptei'  an  account  is  given  of  the 
several  parts  of  the  microscope,  and  the  purpose  for  which 


vi  PEACTICAL  HISTOLOGY 

they  are  intended  (without  entering  into  an  explanation 
of  its  optical  construction),  and  of  the  instruments  and 
the  methods  of  staining  and  otherwise  preparing  tissues 
which  are  in  more  general  use  in  Histologj'.  Here 
also  instructions  will  be  found  for  measuring  and  for 
delineating  microscopic  objects.  The  more  special  methods 
of  preparation  are  given  with  the  tissues  and  organs  for 
which  they  are  employed. 

Throughout  the  book  detailed  descriptions  of  tissues 
have  been  avoided,  seeing  that  these  are  to  be  found  in 
systematic  works,  such  as  Quain's  '  Anatomy,'  Jvlein's 
'  Elements  of  Histology,'  or  the  author's  '  Essentials  of 
Histology.' 

University  College,  London  : 
May  1897. 


CONTENTS 


CHAPTER  PAGE 

Introductory  :  Instruments  and  General  Methods  used 

IN  Histology 1 

I.     The  Blood -.48 

II.     The  Epithelial  Tissues 82 

III.  Connective  Tissue 96 

IV.  Cartilage 114 

V.     Bone      .         .         .         .        , 123 

VI.     Muscular  Tissue 134 

VII.     Nervous  Tissue 146 

VIII.     The  Blood-vessels 162 

IX.    Lymphatics   and    Serous  Membranes.      Synovlil  Mem- 
branes   ......••-•  186 

X.     The  Skin,  Hairs,  and  Nails 199 

XI.     The  Heart 205 

XII.     The  Lungs 208 

XIII.  The  Mouth  and  Pharynx.     Teeth.    Tongue.     Salivary 

Glands 213 

XIV.  The  (Esophagus  and  Stomach 220 

XV.     The  Small  and  Large  Intestines 225 

XVI.     The  Liver  and  Pancreas 231 

XVII.     The  Ductless  Glands 236 


viii  PEACTICAL  HISTOLOGY 

CHAP'J'ER  PAGE 

XVIII.  The  Ukinaby  Organs 242 

XIX.  The  Generative  Organs 247 

XX.  The  Central  Nervous  System 252 

XXI.  The  Organs  of  the  Senses — the  Eye    ....  257 

XXII.  The  Auditory,  Olfactory,  and  Gustatory  Organs    .     .  283 

INDEX 291 


,>*s 


li* 


LIST    OF   ILLUSTEATIONS 


FIG.  PAGE 

1.  Diagram  op  Microscope 2 

2.  Iris  Diaphragm 3 

3.  Sub-stage  Condenser 3 

4.  Simple  Microscope     .........  6 

5.  Compound  Erecting  Microscope  for  Dissecting  ...  7 

6.  Glass  Slide  and  Cover-glass 8 

7.  Forceps  for  taking  up  Cover-glass 9 

8.  Clip  to  hold  Cover-glass 9 

9.  Spear-headed  Needle     . 9 

10.  Scissors,  Forceps,  and  Mounted  Needle          .         .         .     .  10 

11.  Section-lifters 12 

12.  Mode  of  using  Pipette  for  Iekigation 13 

13.  Card  Tray  foe  holding  Microscope  Slides  .         .         .13 

14.  Bottle  for  Xylol  Balsam "25 

15.  Outline  of  Paper  for  Embedding  Trough  .         .         .         .2(5 

16.  Paper  folded  to  forbi  Trough 27 

17.  Embedding  Trough  of  Le.U)  Foil          .....  27 

18.  Section -cutting  with  the  Free  BLod 28 

19.  Freezing  Microtome  (Ether  Spray),  Jung's         .         .         .  29 

20.  Brass  Holders  for  Collodion  Preparations    .        .        .     .  30 


PEACTICAL  HISTOLOGY 


FIG. 


PAGE 


21.  Inclixed  Plane  Mickotome,  Jung's 31 

22.  L-Shaped  Brass  Blocks  fokiiing  Mould  for  P.\E.\FFrN    .     .  32 
28.    box  for  storing  p.\raffix  blocks 33 

24.  Tripod  Microtojie  (Birch's  Pattern) 34 

25.  Glass  Pot  with  Cover 35 

26.  Rocking  Microtome  (Cambridge  Pattern)         .         .         .     .  35 

27.  Minot's  Microtome          ........  36 

28.  Grooved  Trough  to  hold  Microscope  Slides  .        .        .     .  38 

29.  CA3IERA  Lucid  A,  Zeiss 40 

30.  Camera  Lucida  of  Abbe     ........  41 

31.  Upright  Micko-photographic  Camera 42 

32.  Horizontal  Micro-photographic  Camera 43 

33.  Ocular  Micrometer 4.'» 

34.  Lines  of  Stage   Micrometer  viewed  with  Ocular   Micro- 

meter            ...  46 

35.  Simple  Warm  Stage 53 

36.  Mode  of  using  Simple  Warm  Staoj: 54 

37.  Warji  Stage  with  Gas  Regulator 56 

38.  Box    TO    INCLOSE    Microscope,    with    Gas    Regulator    and 

Thermometer        .........  58 

39.  Simple  Moist  Chamber 62 

40.  Gas  Chamber 63 

41.  Slide  ruled  in  Squares  of  0-1  mji.  each  for  Enumeration 

of  Blood-corpuscles         .......  64 

42.  Oliver's  Apparatus  for  Estimating  the  Nujiber  of  Blood- 

corpuscles    ..........  67 

43.  White  Corpuscles  migrating  from  Clot       ....  71 

44.  Slides   for   the   Passage   of   Electric   Shocks   through  a 

Preparation 72 

45.  Apparatus   for  Passing  Electric   Shocks  through  a  Pre- 

pabatiok    . 73 


LIST  OF  ILLUSTKATIONS  xi 

riG.  PAGK 

46.  Appaeatus  for  Passing  CO.,  over  a  Preparation     .        .     .  76 

47.  Valve  of  Mussel,  showing  Gills  ......  90 

48.  Apparatus  foe  Passing  CO.^ 93 

49.  Syringe  foe  Interstitial  Injection 101 

50.  Warming  App.aratus  with  Gas  Eegulatoe        .        .        .     .  119 

51.  Frog-coek  for  viewing  Circulation  in  Web,  Tongue,  oe 

Mesenteey        .        .        .        .    # 171 

52.  Steuctuee  and  Position  of  Tongue  of  Toad  .         ...  174 

53.  Injecting  Apparatus  for  Blood-vessels        ....  180 

54.  Cannulas  and  Clips  used  in  Injecting 182 

55.  Hoggan's  Eings 189 

56.  Mercury-peessuee  Apparatus  foe  Injecting  Lymphatics     .  193 

57.  Fine  Perforated  Steel  Needle  foe  Injecting  Lymphatics  194 

58.  Mode  of  cutting  Feog's  Coenea  to  cause  it  to  lie  flat  .  264 

59.  Tongue  of  Kabbit •  289 


f  coitxtl  fit  Hl^ 


PEACTICAL     HISTOLOGY 


INTRODUCTORY 

INSTRUMENTS    USED    IN    HISTOLOGY 

The  Microscope. — The  practical  study  of  Histology  is 
mainly  dependent  upon  the  use  of  the  microscope.  The  micro- 
scope is  a  combination  of  lenses  arranged  for  the  purpose  of 
obtaining  and  viewing  a  magnified  image  of  any  minute  object. 
The  lenses  are  set  in  a  tube  of  variable  length — the  tube  of  the 
microscope  (fig.  1,  t,  t') — and  this  is  itself  supported  in  a  ver- 
tical position,  on  a  firm  metal  stand,  which  is  provided  with 
an  arrangement  by  which  the  tube  is  capable  of  being  moved, 
without  lateral  deviation,  in  a  perfectly  straight,  up-and-down 
direction.  This  arrangement  is  termed  the  adjustment.  Its 
purpose  is  to  bring  the  microscope  into  that  position  with 
regard  to  the  object  in  which  the  latter  is  most  clearly  seen. 
The  object  is  then  said  to  be  in  focus. 

Two  adjustments  are  commonly  provided  :  one — the  coarse 
adjustment  (adj) — serves  to  bring  the  lenses  roughly  into  the 
focal  position,  and  is  either  a  telescopic  joint  or  a  rack  and  pinion 
movement ;  the  other — the  Jine  adjustment  {adj') — is  a  fine 
screw,  and  by  its  means  the  focus  may  be  obtained  with 
complete  exactness  even  when  the  highest  magnifying  powers 
are  employed.  The  stand  is  further  provided  with  a  rigidly 
connected,  horizontal  table  or  stage  (st),  upon  which  the  object 
is  placed,  and  which  projects  below  the  tube,  and  is  provided 


PEACTICAL  HISTOLOaY 


Fig.  1 


Diagram  of  microscope 
■t,  tube  seen  in  section .;  t',  sUding  part  of  tube  ;  oc,  ocular;  with  .,  eye-glass  and  f  field 

pnragm ,  m,  nurroi- ;  ft,  foot  of  microscope  i       ^    >  i 

The  object  is  indicatea  by  a  small  ai-row  just  above  the  aperture  in  the  dianhra^  •  tbP 
magnified  image  by  the  larger  arrow  in  the  middle  of  the  ocul7      ^ ' 


THE   MICROSCOPE 


3 


with  a  circular  aperture  to  Jidmit  light  from  l)eIo\v  to  the 
object,  capable  of  being  varied  by  means  of  an  iris-diaphrngin 
(fig.  2),  or  hy  stops  (tig.  1,  c/)  furnished  with  holes  of  different  sizes. 
Diffused  daylight  is,  if  possible,  employed,  and  is  i-eflected,  by 
meansof  a  movable  mirror[m)  belowthe  stage,  upwards  through 
the  object  and  through  the  tube  of  the  microscope  to  the  eye 
of  the  observer.  This  is  termed  viewing  an  object  by  trans- 
mitted light.  Occasionally,  especially  when  comparatively 
large  and  opaque  objects  and  low  magnifying  powers  are  to  be 
employed,  the  former  are  viewed  by  the  light  which  is  re- 


Iris-diaphragm 


Substage  condenser 


fleeted  from  their  surface,  whilst  that  from  the  mirror  is  cut 
off.  In  order  in  such  cases  to  concentrate  as  much  light  as 
possible  upon  the  object  a  bull's-eye  condenser  is  employed. 
It  is  only  in  viewing  such  preparations  that  the  binocular 
microscope  offers  any  material  advantage  in  histology. 

To  obtain  a  more  central  and  concentrated  illumination 
for  objects  which  are  to  be  viewed  by  transmitted  light,  a 
condenser  is  frequently  fitted  below  the  stage  of  the  micro- 
scope {substage  condenser,  fig.  .3).  The  stage  may  conveniently 
be  ruled  at  one  part  with  numbered  lines  crossing  one 
another  at  right  angles.     By  this  means  the  position  of  a  slide 

b2 


4  PEACTICAL  HISTOLOGY 

can  be  recorded,  and  an  object  readily  found  again   by  re- 
placing the  slide  in  that  position. 

The  lenses  form  the  essential  part  of  the  microscope,  and 
are  so  arranged  that  one  combination  of  them  is  placed  at  the 
lower  end  of  the  tube  and  produces  a  magnified,  inverted  image 
of  the  object  at  the  upper  part.  The  image  thus  produced 
is  viewed,  and  at  the  same  time  still  further  magnified,  by 
another  combination  at  the  top  of  the  microscope  tube.  The 
lenses  at  the  lower  end  of  the  tube  form  an  achromatic  com- 
bination which,  from  its  situation  near  the  object,  is  termed 
the  object-glass  or  objective,  and  it  is  upon  the  perfection  of  its 
construction  that  the  usefulness  of  the  microscope  mainly  de- 
pends. The  combination  which  is  at  the  upper  end  of  the  micro- 
scope tube  is  termed  the  eye-jnece  or  ocular,  and  consists  of  a 
lens,  the  eye-piece  proper  (e),  placed  close  to  the  eye  of  the  ob- 
server, and  of  another  glass  {t\xG field-glass,  f)  situate  below  the 
first  and  having  for  its  object  the  collection  of  the  more  diver- 
gent rays  transmitted  by  the  objective,  and  also  the  lessening  of 
chromatic  aberration.  The  whole  ocular  (oc)  thus  composed 
is  made  to  slip  in  at  the  superior  aperture  of  the  microscope 
tube.  It  is  seldom  necessary  to  have  more  than  one  ocular,  of 
medium  strength,  in  use,  but  at  least  two  objectives  of  difierent 
magnifying  powers  are  essential  for  ordinary  histological  work. 
One  of  these,  which  in  subsequent  pages  will  be  spoken  of 
as  the  loio  power,  should,  when  used  with  the  ordinary  eye- 
piece, give  an  apparent  linear  enlargement  of  about  75 
diameters  ;  that  is  to  say,  when  a  line,  the  length  of  which 
is  known  (say  -j-w  ^f  an  inch),  is  observed  through  this 
combination  it  should  appear  seventy-five  times  as  long  as 
it  really  is  (|  inch  therefore).  The  other,  to  be  mentioned 
as  the  high  power,  should  given  an  apparent  linear  magnification 
of  from  300  to  400  diameters.  These  two  glasses  amply  suffice 
for  all  ordinary  histological  studies  ;  but  for  certain  special 
subjects  it  is  advantageous  to  obtain  the  use  of  a  more 
powerful  combination — one  that  will  magnify  1,000  diameters 
or  more. 


THE   MICROSCOPE  5 

Glasses  of  this  liigli  maj^nifying  power  are  usually  of  the  kind 
known  as  '  immersion-objectives,'  so  called  because  they  are 
corrected  for  use  with  u  stratum  of  distilled  water  or  of  cedar-oil 
between  the  specimen  and  the  lower  lens  of  the  objective.  It  is 
best  to  apply  tlie  water  or  oil  to  this  lens  with  a  splinter  of  wood 
or  from  a  pipette  before  the  objective  is  screwed  on  to  the  tube  of 
the  microscope ;  the  tube  is  then  lowered  by  means  of  the  coarse 
adjustment  until  the  drop  of  Uqiiid  comes  in  contact  with  the 
cover-glass,  after  which  the  focus  is  obtained  by  cautiously  lower- 
ing it  further,  at  first  with  the  coarse,  and  then  with  the  fine  ad- 
justment. 

Apocliromatic  objectives  (with  compensating  oculars),  which 
were  introduced  by  Abbe  and  Zeiss,  are  now  furnished  by  all  good 
microscoi^e  makers.  They  are  distinctly  superior  to  the  ordinaiy 
objectives. 

The  above  comprise  the  essential  I'equirements  of  a  micro- 
scope, but  larger  instruments  are  often  provided  Avith  certain 
adjuncts  which  render  them  in  a  measure  more  complete. 
The  stand  is  usually  hinged,  so  that  the  stage  and  tube  can 
be  tilted  somewhat  out  of  the  perpendicular  to  allow  of  better 
adaptation  to  the  position  of  the  observer.  But  if  the  micro- 
scope tube  is  not  inconveniently  high,  it  is  almost  as  comfoi't- 
able  to  work  without  inclining  the  instrument,  and  some 
preparations,  those,  for  instance,  which  have  to  be  examined 
in  fluid,  will  not  admit  of  inclination.  A  mechanical  stag/', 
i.e.  a  stage  capable  of  carrying  the  object  under  examination 
horizontally  in  two  directions  by  screw  adjustment  or  other 
mechanical  means,  instead  of  by  hand,  is  sometimes  added,  but 
for  most  work  can  very  well  be  dispensed  with.  A  camera 
hicidd  (see  p.  41)  is  useful  for  obtaining  an  exact  sketch  of 
the  outlines  of  an  object.  A.  nose-piece,  holding  two  or  even 
three  or  more  objectives,  which  can  be  changed  by  a  simple 
act  of  rotation,  is  a  very  convenient  adjunct. 

A  polarisation  apparatus  is  occasionally  employed  in 
investigating  the  optical  properties  of  the  substances  which 
compose  the  tissues,  and  is  also  of  use  in  helping  to  determine 
the  nature  of  crystalline  deposits  in  urine  and  other  fluids. 


6 


PKACTICAL  HISTOLOGY 


In  connection  with  the  employment  of  this  the  stage-plate 
of  the  microscope  is,  in  the  best  instruments,  made  capable  of 
rotating  on  a  vertical  axis.  In  smaller  instruments  this  move- 
ment is,  as  a  rule,  not  provided  for,  and,  indeed,  although 
convenient,  is  not  essential,  ^ 

Perfect  steadiness  of  the  stand,  stage,  and  adjustments  is 
the  most  important  point  to  pay  attention  to  in  selecting  a 

Fig.  4 


Simple  form  of  dissecting  microscope,  provided  with  tliree  lenses  of  different 
magnifying  power 


useful  microscope,  so  far  as  the  body  of  the  instrument  is  con- 
cerned. The  excellence  of  the  objectives  can  only  be  compe- 
tently judged  of  by  one  who  is  already  somewhat  conversant 
with  the  use  of  the  microscope. 

In  addition  to  the  ordinary  instrument  (which  is  generally 

1  A  description  of  the  method  of  using  the  polarisation  apparatus  is  given 
in  the  chapter  on  muscle  (p.  142). 


INSTJiUMENTS 


distinguished  as  the  '  compound  '  microscope),  it  \v\\\  be  found 
very  convenient  to  have  a  simple  microscope  as  well  :  one  is 
thus  better  able  to  follow  the  needles  or  other  instruments 
when  engaged  in  the  separation  and  manipulation  of  minute 
objects.  An  instrument  which  is  used  for  this  purpose  is 
termed  a  dissecting  or  prejmriiuj  microscope.  Any  simple 
lens  which  is  mounted  on  a  stand  will  serve  (fig.  4),  and  even 
the  bull's-eye  condenser,  Avhich  is  frequently  furnished  with  the 
microscope,  may  be  employed  as  a  lens  if  the  marginal  part  is 

Fi(i.  .-. 


Compound  dissecting  microscope,  nmde  by  Xachet 

covered  by  a  black-paper  diaphragm.  A  very  convenient 
form  of  dissecting  microscope  is  shown  in  fig.  5.  Tliis  con- 
sists of  a  small  compound  microscope  with  a  low-power  objec- 
tive and  furnished  with  an  ocular  constructed  for  reversing 
the  image,  so  that  the  object  appears  in  its  natural  positioii 
and  not  inverted.  It  is  provided  also  with  a  prism  for 
reflecting  the  light  in  a  direction  convenient  for  the  eye,  and 
is  placed  on  a  wooden  stand,  so  constructed  as  to  afford  support 
to  the  arms  of  the  operator. 

Instruments. — Besides  the  microscope,  the  student  wluo  is 
commencing   the   practical   study   of   histology    will    tind   it 


8 


PEACTICAL  HISTOLOGY 


necessary  to  be  provided  with  the  following  simple  instruments 
and  appliances  : — 

Glass-slides  and  cover-glasses  (see  fig.  6). — Microscopic 
slides  are  oblong  slips  of  glass  upon  which  the  object  is  placed. 
In  this  country  they  are  usually  cut  of  the  uniform  and  con- 
venient size  of  three  inches  by  one  inch.  The  glass  should  be 
quite  free  from  flaws  and  specks.  The  cover-glasses  should  be 
the  finest  sold.  It  is  true  that  when  very  delicate  they  are 
likely  to  be  broken  in  the  cleaning  unless  great  care  is 
exercised  ;  but,  on  the  other  hand,  some  high-power  objectives 
require  to  be  focussed  so  close  to  the  object  that  a  thick  cover- 

FiG.  6 


Glass  slide  and  cover-glass,  natural  size 

The  figure  shows  the  mode  of  letting  down  the  covei;-glass  (with  a  drop  of  fluid  on  its 
under  surface)  gentlj'  on  to  the  middle  of  the  slide 

glass  cannot  be  used  without  risk  of  crushing  the  tissue,  and 
perhaps  of  scratching  the  objective.  As  a  rule,  the  chief 
difficulty  in  cleaning  cover-glasses  arises  from  the  fact  that 
their  surfaces  are  often  covered  with  a  thin  film  of  grease  or 
other  organic  matter  which  it  is  almost  impossible  to  rub  off. 
But  this  is  easily  got  rid  of  by  placing  a  number  of  them 
together  in  a  small  glass  beaker  and  pouring  a  little  strong- 
nitric  acid  upon  them  :  this  quickly  destroys  every  trace  of 
organic  matter.  The  acid  is  then  poured  ofi",  and  the  cover- 
glasses  are  thoroughly  rinsed  by  allowing  water  to  stream 
over  them  from  a  tap  for  two  or  three  minutes.  They  may 
then  be  kept  in  distilled   water  or  spirit  ready  for  use,  and 


INSTliUMKNTS 


need  only  be  dried  when  wanted.  The  drying  is  effected  by  a 
thin  linen  or  cotton  cloth,  which  is  laid  flat  on  the  table, 
and  the  cover-glass,  having  been  placed  on  this,  is  gently 
rubbed,  first  on  the  one  surface  and  then  on  the  other,  with 
a  corner  of  the  cloth.  Either  ordinary  or  special  forceps 
(fig.  7)  may    be  used  for  placing  the  cover-glass  in  position 

Fig.  9 


Fig. 


Fig.  8 


Cover-glass  forceps        Cover-glass  holder  (Zoth)       Spear-headed  needle 

on  the  slide,  but,  as  a  rule,  an  expert  microscopist  can  perform 
the  operation  equally  well  with  the  cover-glass  held  between 
the  thumb  and  forefinger  applied  to  opposite  edges.  When 
cleaned  and  dried  ready  for  use  the  cover- glass  may  be  held  in 
a  small  metallic  clip  (fig.  8).  Square  cover-glasses  should 
always  be  used  in  preference  to  round  ones  when  reagents 
have  to  be  applied  to  a  specimen  under  the  microscope.     The 


10 


PKACTICAL  HISTOLOGY 


Fig.  10 


Scissors  and  forceps  suitable  for  liistological  purposes.     Natural  size 
n,  eud  of  a  mounted  needle 


INSTRUMENTS  1 1 

most  convenient  size  for  general  purposes  is  '{  inch  (about 
2  centimetres)  square. 

Mounted  needles. — These  are  sewing-needles  mounted  in  a 
wooden  handle,  with  about  ^  inch  (1'5  centimetres)  of  their 
pointed  end  projecting  (fig.  10,  n).  They  are  amongst  the  most 
useful  instruments  which  the  histologist  possesses,  and  will  be 
in  constant  requisition.  They  must  always  be  kept  clean  and 
sharp.  A  spear-headed  needle  is  also  frequently  a  useful 
adjunct  (tig.  9). 

Scissors  and  forceps. — A  small  pair  of  scissors  with  short, 
straight  blades  is  necessary.  Their  cutting  edge  must  always 
be  kept  very  sharp,  especially  towards  the  point.  A  small 
curved  pair  is  often  useful.  At  least  two  pairs  of  small  steel 
forceps  are  requisite.  The  blades  should  be  short,  broad  at 
their  junction,  so  as  not  to  admit  of  lateral  deviation,  and 
tapering  to  a  blunt  point.  They  are  slightly  roughened  at 
the  end,  so  as  to  afford  a  firmer  grasp. 

Section-lifters. — One  or  two  section-lifters  (tig.  11)  of  differ- 
ent sizes  are  required.  They  are  made  of  platinum,  steel,  copper, 
or  German  silver,  and  may  be  either  flat  or  slightly  concave, 
in  the  latter  case  with  a  small  hole  in  the  centre. 

Slips  of  loJiife  blotting  paj)er  should  always  be  at  hand. 
They  serve  both  for  soaking  up  excess  of  fluid  from  under  the 
cover-glass  and  for  placing  the  slide  upon,  when  preparing  a 
tissue  that  has  been  stained,  so  that  it  is  better  seen  than  it 
would  be  upon  a  black  surface.  On  the  other  hand,  it  is 
better  to  use  a  black  surface  for  working  upon  when  tissues 
are  unstained.  A  numljer  of  shallow  glasx  or  porcelain  pots 
provided  with  well-fitting  covers  (see  fig.  25),  and  one  or 
two  large  shalloio  glass  or  porcelain  dishes  (such  as  are  used 
by  photographers)  are  in  constant  requisition,  since  very 
many  of  the  manipulations  require  to  be  performed  whilst  tlie 
tissue  is  immersed  and  floated  out  in  fluid  :  while  for  applying 
reagents  to  a  specimen  under  the  microscope  small  pipettes 
(fig.  12)  are  extremely  convenient.  Pipettes  may  be  readily 
made  with  the  aid  of  a  blow-pipe  by  drawing  out  a  piece  of  soft 


12 


PKACTICAL  HISTOLOGY 
Fig.  11 


Section-lifters.     Natural  size 


INSTRUMENTS 
Fig.  12 


13 


Method  of  irrigating  a  preparation  with  fluid  from  a  capihary  pipette 
i>,  blotting  paper 

Fig.  13 


Card  tray  for  holding  twenty  microscope  slides 


14  '       PRACTICAL  HISTOLOGY 

glass  tube  at  two  places  close  to  one  another,  so  that  the  inter- 
mediate part  remains  as  the  bulb  of  the  pipette.  It  is  well  to 
make  a  number  at  a  time,  sealing  up  the  ends  in  the  flame 
while  the  bulb  is  still  hot ;  they  are  thus  sterilised  and  made 
dust-tight,  and  may  be  kept  always  ready  for  use.  It  is  only 
necessary  to  break  off  the  sealed  ends  when  required  and  to 
suck  the  re-agent  up  into  the  bulb.  A  pipette  should  always 
be  rejected  after  it  has  once  been  used,  and  never  employed 
for  another  reagent. 

For  keeping  preparations  that  have  been  permanently 
mounted,  flat  trays  to  hold  twenty  or  more  specimens  are  the 
most  useful  (tig.  1 3).  They  are  conveniently  made  of  cardboard 
or  2^<:i'P'ier  mdche  with  the  sides  folding  over  the  middle,  so  that 
the  preparations  are  completely  shielded  from  dust,  and  may 
thus  be  arranged  on  shelves  or  in  cabinets.  Boxes  with  grooved 
sides  in  which  the  specimens  stand  vertically  are  also  very 
convenient  and  of  less  cost. 

REAGENTS    USED    IN    HISTOLOGY 

A  vast  number  of  reagents  are  from  time  to  time  and  for 
various  purposes  employed  in  special  histological  work,  but 
comparatively  few  are  needed  for  ordinary  work.  These, 
therefore,  will  alone  be  mentioned  in  this  place,  others  being 
referred  to  as  they  are  required  for  any  particular  object. 

SOLUTIONS    FOR    THE    EXAMINATION    OP    THE    FRESH    TISSUES 

Serum  of  blood  and  lymph  form  the  most  natural  fluids  in 
which  to  prepare  fresh  tissues  for  examination.  As  a  form  of 
lymph,  the  aqueous  humour  of  the  eye  may  be  also  employed, 
and  has  the  advantage  that  it  can  always  easily  be  got  from  a 
recently  killed  animal.  But  the  albuminous  nature  of  these 
fluids  is  to  this  extent  an  objection  to  their  use,  that  it  prevents 
the  ready  employment  of  hardening  or  fixing  reagents  if  it 
should  be  necessary  afterwards  to  use  these.  This  objection 
does  not  apply  to  salt  solution,  which  in  the  form  of  the  so- 


FIXATJVE   SOLITJONS  15 

called  normal  saline  solution  (common  salt  G  to  9  parts,  tap- 
water  1,000  parts)  is  extensively  used.  It  must  be  under- 
stood that  a  strength  of  salt  solution  which  is  noimal  for  one 
animal  may  not  be  so  for  another.  Thus  a  saline  solution  of 
G  pai'ts  to  1,000,  which  is  normal  or  isotonic  for  frogs'  l)lood- 
corpuscles  {i.e.  tends  to  produce  no  alteration  in  their  form 
by  either  diffusing  into  or  abstracting  water  from  them),  is 
subnormal  (hypotonic)  for  mammalian  corpuscles,  which  require 
9  parts  per  1,000  to  give  a  like  result. 


SOLUTIONS    FOR    FIXING   THE    TISSUES   AND    ORGANS  WITH   THE 
FORM    AND   STRUCTURE    TIIEY    POSSESS    DURING    LIFE 

Many  of  the  most  valuable  solutions  for  this  purpose  are 
of  an  acid  nature.  Those  most  commonly  employed  are 
chromic  acid  and  the  bichromates,  osmic  acid,  acetic  acid, 
picric  acid,  nitric  acid,  corrosive  sublimate,  or  mixtures  of 
two  or  more  of  these  ;  last  but  not  least,  alcohol.  Recently 
also  formaldehyde  has  been  introduced  for  the  same  purpose. 
The  strength  of  the  solutions  used  varies  with  the  pui'pose  for 
which  they  are  to  be  employed.  If  the  elements  of  the  tissue 
are  to  be  dissociated,  weak  solutions  of  the  fixing  fluids  are 
taken  ;  if  its  parts  are  to  be  kept  together  and  the  pieces 
hardened  for  the  preparation  of  sections,  stronger  solutions 
are  employed.  In  every  case  the  fluid  should  far  exceed  in 
bulk  the  piece  of  tissue  or  organ  to  be  preserved,  and  the 
latter  should  never  be  too  thick  for  the  preserving  fluid  readily 
to  penetrate  to  eveiy  part.  It  is  best,  if  possible,  to  inject  it 
into  the  blood-vessels  or  into  the  interstices  of  a  tissue. 

Chromic  acid. — This  may  be  kept  as  a  1  per  cent,  solution, 
to  be  diluted  as  required.  Chromic  acid  is  an  invaluable  re- 
agent for  fixing  and  hardening,  and  for  this  purpose  a  strength 
of  solution  varying  from  0*2  per  cent,  to  1  per  cent,  may  be 
used.  It  is  best,  as  a  rule,  to  use  the  weaker  solutions,  since 
the  stronger  are  liable  to  interfere  with  the  staining  of  the 
preparations  by  dyes.     Tissues  are  hai'dened  by  chromic  acid 


16  PEACTICAL  HISTOLOGY 

in  a  few  days.  For  use  as  a  dissociant  the  solution  may  be 
diluted  down  to  0-05  per  cent.  (J^  per  cent.)  or  more. 

Bichromates. — Bichromate  of  potash  and  bichromate  of 
ammonia  are  both  used  extensively  in  histology,  in  solutions 
of  2  per  cent,  to  3  per  cent.  They  have  the  advantage  over 
chromic  acid  that  they  penetrate  a  piece  of  tissue  more  rapidly, 
so  that  larger  portions  of  organs  can  be  hardened  in  them,  and 
they  interfere  less  than  the  acid  with  the  subsequent  staining 
by  dyes.  But  they  do  not  preserve  the  minute  structure  of 
the  tissues,  at  least  of  their  cells  and  nuclei,  as  do  solutions  of 
chromic  acid  ;  nor  is  the  hardening  process  so  rapid  (2  to  4 
weeks).  Muller's  fluid  is  a  2^  per  cent,  solution  of  bichromate 
of  potash  in  water  containing  1  per  cent,  of  sulphate  of  soda. 

Osmic  acid. — This  is  the  most  rapid  fixative  for  most  tissues, 
and  in  some  respects  the  most  perfect,  for  it  usually  kills 
protoplasmic  structures  so  suddenly  that  there  is  no  time  for 
any  contraction  or  other  change  of  form  to  occur.  On  this 
account  it  is  extensively  used,  alone  and  combined  with  other 
reagents.  But  it  has  the  disadvantage  that,  more  than  most 
other  fixatives,  it  interferes  with  the  subsequent  staining  of 
cells  by  dyes  ;  nor  does  it  bring  out  the  fine  structure  of  cells 
and  nuclei  as  well  as  chromic  or  picric  acid.  It  stains  sub- 
stances of  a  fatty  nature  black.  It  is  usually  allowed  to  act 
on  a  tissue  only  for  a  short  time  (15  minutes  to  3  hours).  It 
is  conveniently  kept  as  a  1  per  cent,  or  2  per  cent,  solution. 

Acetic  acid. — This  is  rarely  used  alone  as  a  fixative,  but 
has  been  a  good  deal  employed  in  combination  with  other 
reagents.  Acetic  acid  is  also  useful  for  determining  the 
action  of  weak  acids  upon  the  tissues.  For  this  purpose  it 
may  be  kept  in  1  per  cent,  solution. 

Flemming's  fluid  is  constituted  as  follows  : — 

Osmic  acid,  2  per  cent.       .         .       4  parts 
Chromic  acid,  1  per  cent.  .         .     15     „ 
Glacial  acetic  acid      .         .         .       1     „ " 

Hermann's  fluid  has  a  similar  composition,  but  with 
1  per  cent,  platinic  chloride  substituted  for  the  chromic  acid. 


FIXATIVE   SOLUTIONS  17 

These  solutions  may  be  used  undiluted,  or  they  may  be 
diluted  up  to  ten  or  even  twenty  times  with  water.  Tissues 
are  hardened  rapidly  in  them  (2  to  5  days). 

Picric  acid. — One  of  the  most  valuable  fixatives.  It  is 
used  in  saturatetl  or  half-saturated  solution,  either  alone  or 
with  the  addition  of  sulphuric  or  nitric  acid  to  the  solution  in 
the  proportion  of  2  p.c.  It  hardens  (juickly,  a  day  or  two 
being  usually  sutHcient.  Its  chief  disadvantage  is  thediHiculty 
of  getting  rid  of  the  excess  of  acid  from  the  tissue  (this  being 
inimical  to  the  after-staining  with  dyes).  With  this  object 
a  tissue  hardened  in  picric  acid  is  placed  for  some  days  in 
95  p.c.  alcohol  containing  lithium  carbonate  in  solution,  the 
fluid  being  changed  until  the  tissue  yields  no  more  colour  to 
it.  The  tissue  is  then  kept  in  alcohol.  It  should  not  bpi 
placed  in  water  before  alcohol. 

Alcohol.-  -This  is  used  in  histology  more  than  any  other 
reagent.  In  strength  of  70  per  cent,  upwards  it  serves 
both  for  fixing  and  hardening,  the  latter  process  being 
extremely  speedy,  especially  when  the  alcohol  is  absolute.  In 
a  strength  of  33  per  cent,  (one-third  alcohol),  as  recommended 
by  Ranvier,  it  is  an  invaluable  dissociant.  It  has  the  great 
advantage  that  it  rather  assists  than  prevents  the  after-stain- 
ing by  dyes.  It  tends  to  produce  a  certain  amount  of  shrinkage 
in  some  of  the  tissue  elements,  so  that  their  intimate  structure 
is  as  a  rule  less  well  displayed  than  after  chromic  or  picric 
acid.  But  alcohol  is  almost  invariably  used  to  complete  the 
hardening  process  Avhen  these  acids  have  acted  sufficiently 
long  to  fix  the  tissue  elements,  and  it  is  also  of  universal 
employment  for  dehydrating  preparations  which  have  to  be 
transferred  from  watery  solutions  to  essential  oils.  The  most 
useful  strengths  of  alcohol  to  keep  ready  are  33  per  cent.,  50  per 
cent.,  75  per  cent.,  96  per  cent,  (or  methylated  spirit),  and 
absolute  alcohol. 

Formaldehyde  has  of  late  come  into  use  as  a  fixing  and 
hardening  reagent.  It  is  sold  in  a  iO  per  cent,  solution, 
which  is  termed /urmoJ.    It  acts  rapidly  and  penetrates  freely, 

c 


18  PEACTICAL  HISTOLOaY 

so  that  a  small  piece  of  tissue  may  be  hardened  in  it  in  the 
course  of  a  few  minutes,  and  then  transferred  to  alcohol. 
Formol  is  rarely  used  undiluted.  For  fixing  and  hardening 
purposes  it  is  used  in  a  strength  of  from  1  in  20  to  1  in  5, 
according  to  the  nature  and  thickness  of  the  tissue.  It  may 
be  mixed  either  with  water  or  with  alcohol.  Tissues  as  a  rule 
stain  well  after  formol,  and  its  rapidity  of  action  and  pene- 
trating power  render  it  a  most  valuable  reagent. 

Corrosive  sublimate  is  best  used  dissolved  in  1  per  cent, 
salt  solution,  as  a  saturated  solution  (which  should  be  kept  in 
the  dark).  It  may  be  used  either  alone  or  with  1  g.  picric 
acid  added  to  each  100  c.c.  of  the  sublimate  solution.  Mann 
further  adds  2  g.  tannic  acid.  Objects  are  fixed  and  hardened 
quickly,  small  objects  in  a  few  minutes,  larger  ones  in  a  few 
hours.  They  are  then  transferred  to  75  p.c.  spirit  deeply 
coloured  by  tincture  of  iodine,  and  after  remaining  in  this 
long  enough  to  remove  the  excess  of  sublimate  they  are 
transferred  to  stronger  spirit,  which  should  be  changed  once 
or  twice.  Objects  hardened  in  corrosive  sublimate  usually 
stain  readily  with  most  dyes.  If  the  blood-vessels  are  in- 
jected with  it,  after  being  washed  clear  of  blood  by  normal 
salt  solution,  fixation  and  hardening  is  instantaneous.  The 
tissues  can  then  be  placed  in  a  quantity  of  the  same  fluid  and 
transferred  in  a  few  hours  to  rectified  spirit  containing  iodine. 
They  are  then  passed  through  stronger  alcohol  to  absolute. 

fluids'  used  for  staining  histological  objects  ' 

A  piece  of  a  tissue  or  organ  may,  after  having  been  fixed 
and  hardened,  either  be  stained  in  bulk,  or  teased  preparations 
or  sections  may  first  be  made  from  it,  and  these  may  then  be 
subjected  to  the  action  of  a  staining  fluid.  Bulk  staining 
usually  requires  several  hours'  immersion  in  a  weak  solution 
of  a  dye,  but  sections  may  usually  be  stained  in  a  few  minutes 

1  Fluids  and  methods  of  staining  for  special  purposes  are  given  in  the 
descriptions  of  the  preparation  of  the  several  tissues  and  organs. 


STAINING  FLUIDS  19 

in  stronger  solutions.       The  following  staining  Huids  are   in 
most  frequent  use  : — 

Haemalum  (Mayer). — This  is  a  solution  of  luoniatein — the 
colouiing  principle  of  h;vmatoxylin  ' — in  alcohol,  added  to  a 
solution  of  aluin  in  water. 

Ammouia  alnin    .     ~)0  grammes        Htematem    .         .      1   gramme 
Distilled  water     .     1,000  c.c.  Ilectitieel  spirit     .      100  c.c. 

Pour  the  h;ematein  solution  gradually  into  the  alum  solu- 
tion. Add  a  small  piece  of  thymol  to  prevent  the  growth  of 
moulds.  This  is  the  stock  solution.  It  may  be  used  in  full 
strength  when  rapid  staining  or  overstaining  is  desired,  or  it 
may  be  diluted  to  any  degree  with  distilled  water.  It  forms 
the  most  generally  useful  form  of  stain  for  sections,  and  is 
in  constant  requisition. 

Delafield's  haematoxylin. — Dissolve  4  g.  hsematoxylin  in 
25  c.c.  absolute  alcohol,  and  add  the  solution  to  400  c.c.  of  a 
concentrated  solution  of  ammonia  alum  in  distilled  water. 
Allow  the  mixture,  which  assumes  a  deep  violet  colour,  to 
stand  3  or  4  days,  and  then  add  100  c.c.  of  glycerine  and 
100  c.c.  of  methylic  alcohol.  The  solution  must  be  allowed  to 
stand  a  few  days  before  it  will  stain  readily,  but  the  results 
which  are  then  obtained  are  extremely  good.  Both  this 
solution  and  Mayer's  haemalum  tend  to  lose  their  staining 
properties  when  long  kept,  and  acquire  a  red  colour,  but  their 
original  colour  and  staining  power  may  be  restored  by  the 
cautious  addition  of  ammonium  sulphide.  They  should  always 
be  filtered  before  using,  and  diluted  with  distilled  water  only. 

Acid  haematoxylin  (Kulschitsky). — Dissolve  1  gramme 
haematoxylin  in  a  little  alcohol,  and  add  to  it  100  c.c.  of  a  2 
per  cent,  solution  of  acetic  acid  in  water.  Preparations  after 
immersion  in  this  fluid  must  be  well  washed  with  tap-water 
or  lithium  carbonate  solution  until  the  brown  coloration  is 
replaced  by  blue. 

1  In  the  following  text  the  terms  logwood  solution,  hajmatosylin,  or 
hsematein,  are  used  indifferently  for  the  colouring  principle  of  logwood. 


20  PEACTICAL  HISTOLOGY 

A  similar  stain  is  obtained  by  adding  2  parts  glacial  acetic 
acid  to  100  parts  hsemalum  (Mayer). 

Kleinenberg's  hsematoxylin. — This  is  useful  for  staining 
in  bulk.     It  is  prepared  as  follows  : — 

(a)  Make  a  saturated  solution  of  crystaUised  calciiun  chloride 
in  70  per  cent,  alcohol  and  add  alum  to  saturation,  (b)  Make  also 
a  saturated  solution  of  alum  in  70  per  cent,  alcohol.  Add  (a)  to  (b) 
in  the  proportion  of  1  :  S.  To  the  mixture  add  a  few  drops  of  a 
saturated  solution  of  hasmatosylin  in  absolute  alcohol. 

It  may,  if  required,  be  diluted  with  the  mixture  of  a  and 
B.     The  stained  tissue  can  be  placed  at  once  in  strong  spirit. 

Heidenhaiii's  bulk  stain. — This  is  one  of  the  best  stams 
for  glandular  organs.  It  is  used  as  follows.  Two  solutions 
are  prepared,  a  and  b  : — 

A  B 

Hsematoxyhn       .  1    gramme  YeUow  chromate 

Distilled  water     .  300   c.c.  of  potash     .        1  gramme 

Water     .         .   200  grammes 

Dissolve  the  hjematoxylin  in  a  little  alcohol  and  add  the 
water.  A  piece  of  tissue  after  hardening  in  alcohol  (or  picric 
acid  followed  by  alcohol)  is  placed  in  the  hiematoxylin  solution 
(a)  for  12  to  '2i  hours,  and  is  then  transferred  for  12  to  2-1 
hours  to  the  chromate  of  potash  solution  (b). 

Carminate  of  ammonia. — Dissolve  one  or  more  grammes 
of  carmine  in  a  small  quantity  of  ammonia.  Place  in  a 
porcelain  capsule  with  a  small  piece  of  thymol  and  aUow 
slowly  to  dry.  Dissolve  in  water  as  required.  Place  the 
tissue  or  sections  in  acidulated  water  after  staining. 

Picrocannine. — Dissolve  1  gramme  of  carminate  of  am- 
monia in  35  c.c.  distilled  water,  and  gradually  add  to  it  with 
constant  agitation  15  c.c.  of  saturated  solution  of  picric  acid. 

Mayer's  carmalnm. — -For  sections  or  bulk-staining. 

Carminic  acid  (Griibler's)       ....      1  gramme 
Alum      ........    10  gi-annnes 

Distilled  water '200  c.c. 

Boil  sogether,  allow  to  cool,  and  filter.     Add  thymol  to  keep. 


STAINING   FLUIDS  21 

This  is  an  invaluable  staining  fluid.  If  sections  which 
have  been  stained  with  it  are  placed  in  alcoholic  solution  of 
picric  acid,  all  the  eflfects  of  picrocarmine  staining  are  obtained. 

Aniline  dyes. — It  may  be  stated,  as  a  general  rule,  appli- 
cable to  all  the  aniline  dyes,  that  the  staining  power  of  a  given 
strength  of  solution  varies  inversely  with  the  amount  of  alco- 
hol present.  Absolute  alcohol  therefore  rapidly  extracts  most 
of  these  dyes  from  preparations  which  have  been  stained  with 
a  watery  or  dilute  alcohol  solution  of  them.  Acid  fuchsin  forms 
an  exception  to  this  rule.  The  aniline  dyes  most  commonly 
employed  are  the  basic  colouring  matters  methyl-green,  ^  rnethyl- 
hlue,  fuchsin  or  magenta,  gentian-violet,  methylene-hlue,-  methyl- 
violet,  safranin,  thionin,  tolicidin-blue,  and  vestivin,  and  the  acid 
colouring  matters  eosin,  acid  fuchsin  (ii-vhin  S),  and  orange  G. 

They  are  best  kept  in  saturated  solutions  in  96  per  cent, 
alcohol  or  in  water.  For  purposes  of  staining,  either  the 
saturated  watery  solution  is  used,  or  a  dilute  solution  is  made 
by  adding  some  of  the  strong  alcoholic  or  watery  solution  drop 
by  drop  to  distilled  water  in  a  watch-glass.  To  obtain  a  very 
intense  stain,  the  following  method  is  used  (Loeffler)  : — Add 
30  c.c.  of  the  saturated  alcoholic  solution  to  100  c.c.  of  a  O'Ol 
per  cent,  solution  of  caustic  potash  in  distilled  water.  Or  : — 
Add  1  c.c.  of  a  1  per  cent,  solution  of  caustic  soda  to  100  c.c. 
aniline  water  (freshly  prepared  by  shaking  up  5  c.c.  aniline 
with  100  c.c.  distilled  water,  and  passing  through  a  filter 
wetted  with  water),  and  in  this  either  dissolve  the  dry  staining 
material  with  repeated  ag-itation  or  simply  add  to  it  some  of 
the  alcohoHc  solution  of  the  dye. 

^  Methyl-green  is  essentially  a  stain  for  nuclear  ekromatin,  and  it  differs 
from  most  other  aniline  dyes  ta  the  fact  that  it  must  always  be  used  in  acid 
solution  (1  p.c.  acetic  acid). 

-  Methylene-blue  is  chiefly  used  in  normal  histology  for  the  staining  of 
nerve-terminations  intra  vitam,  or  at  least  perfectly  fresh.  The  method  of 
employing  it  for  this  purpose  will  be  given  later.  It  can  also  be  used  in 
stronger  solution  to  show  epithelial  outlines,  cell-spaces  of  connective  tissue, 
&c.,  such  as  are  exhibited  by  nitrate  of  silver  (^p.  103).  It  is  fixed  in  the  tissue 
by  subsequent  treatment  with  nitro-m^olybdate  of  ammonia.  Methyl-blue  is 
quite  a  different  substance. 


22  PEACTICAL  HISTOLOGY 

For  karyokinetic  figures  and  centrosomes  of  cells  it  is  best,  as 
recommended  by  Henneguy,  to  place  the  sections  before  staining  in 
1  p.c.  solution  of  permanganate  of  potash  for  5  minutes  (after 
which  they  are  to  be  thoroughly  washed  with  water). 

In  using  most  aniline  dyes  it  is  customary  to  overstain  and 
afterwards  partially  decolourise.  Alcohol  may  be  used  for 
this  purpose,  with  or  without  the  addition  of  O'l  to  2  per 
cent,  hydrochloric  acid.  When  acid  is  used  it  must  always 
be  completely  removed  by  washing  first  with  water  or  80  p.c. 
alcohol  containing  a  little  lithium  carbonate  and  then  ordinary 
water  or  alcohol.  The  process  of  decolourisation  is  arrested  by 
transferring  the  preparations  to  oil  of  cloves,  oil  of  bergamot, 
or  xylol.  Acid  alcohol  is  used  also  to  decolourise  sections 
overstained  by  hsematoxylin.  Alcohol  rendered  alkaline  with 
caustic  potash  is  used  to  decolourise  tissues  overstained  by 
acid  fuchsin. 

Double  and  triple  staining. — Ficrocarmine,  magenta, 
hoi'inalum-eosin,  r)%et1iyl-hlue  eosin,  toluidin-hlue  eosin,  Ehrlich- 
Biondi  stain. — Two  or  three  stains  giving  different  results 
may  be  used  for  a  preparation,  and  they  may  be  applied  suc- 
cessively or  simultaneously.  Ficrocarmine  is  in  fact  such  a 
double  stain,  since  it  dyes  some  tissues  yellow  and  others  red. 
Magenta  (fuchsin)  also  gives  a  double  stain,  some  tissues 
being  coloured  purple  and  others  red.  For  hardened  tissues, 
which  are  to  be  prepared  as  sections  after  staining  in  bulk,  a 
saturated  solution  in  rectified  spirit  may  be  used  (see  Ossifi- 
cation, p.  131).  For  fresh  tissues  the  following  solution  is 
recommended.  Take  '05  gr.  magenta  and  dissolve  it  in  10  c.c. 
of  alcohol.  To  the  solution  add  gradually  20  c.c.  glycerine 
and  then  water  to  100  c.c.  The  fresh  tissue  is  to  be  mounted 
in  this  fluid,  and  the  cover-glass  soon  cemented,  to  prevent 
evaporation  of  water  and  concentration  of  the  glycerine,  which 
would  result  in  the  removal  of  the  stain  from  the  tissue  ele- 
ments.    Elastic  fibres  are  stained  intensely  by  this  means. 

Eosin-hoimatein. — One  of  the  simplest  methods  of  double 
staining  of  sections  is  to  place  them  first  for  20  minutes  in 


STAINING    FLUIDS  2H 

I  percent,  water  solution  of  rosin,  and,  after  washing  with  dis- 
tilled wator,  m  /untKtlun)  until  stained.  Another  way  is  to 
stain  with  lia'niatein  first,  and  afterwards  with  alcoholic 
solution  of  eosin.  From  this  they  are  passed  directly  into 
clo\e  oil.     Oran(/i'  G  may  be  used  in  place  of  eosin. 

Melhyl'hlne  and  rosin. — This  may  be  made  as  follows. 
I  )issolve  methyl-blue  and  eosin  to  saturation  in  water,  filter, 
and  add  a  little  thymol  to  the  solution.  Use  the  stain  either 
as  it  is  or  diluted  with  water  from  5  to  20  times,  according  to 
the  nature  t)f  the  tissue  and  the  manner  in  which  it  has  been 
fixed  and  hardened.  The  time  of  staining  will  also  depend 
upon  these  factors  :  thus,  with  some  sections  half  a  minute  will 
be  enough  ;  with  others,  differently  hardened,  a  much  longer 
time,  even  hours,  may  be  necessary.  The  sections  may  be 
overstained  and  decolourised.  This  last-named  process  is 
effected  by  absolute  alcohol,  or,  more  rapidly,  by  alcohol  con  - 
taining  1  or  2  per  cent.  HCl.  The  sections  are  then  passed 
through  pure  alcohol  into  an  alcoholic  solution  of  eosin  (1  per 
cent.),  where  they  remain  for  a  few  minutes  ;  they  are  then 
ra[)idly  dehydrated  in  absolute  alcohol  and  transferred  to  oil  of 
cloves,  oil  of  bergamot,  or  xylol. 

Tolaidin  hbic.  and  eosin  stain  (Mann). — Place  the  prepara- 
tion for  ten  minutes  in  a  1  per  cent,  water  solution  of  eosin  ; 
rinse  with  water  and  place  for  twenty  minutes  in  a  1  per  cent, 
solution  of  toluidin  blue.  Decolourise  just  sufficiently  and 
dehydrate  in  absolute  alcohol,  transfer  to  xylol  and  mount 
in  xylol  balsam.     This  is  a  very  good  general  method. 

Ehrlicli-Biondi  triple  stain. — This,  as  modified  by  Heiden- 
hain,  is  made  as  follows.  Saturated  aqueous  solutions  of 
orange  C,  acidfuchsin,  and  rnethyl-green  are  prepared,  filtered, 
and  mixed  as  follows  : — To  100  c.c.  of  the  orange  G  solution 
20  c.c.  of  the  acid  fuchsin  is  gradually  added,  and  then  to  the 
mixture  -'lO  c.c.  of  the  methyl-green.  For  actual  staining  this 
solution  may  be  diluted  with  water  up  to  100  times  its  volume. 
Before  being  used  it  should  be  faintly  acidulated  with  acetic 
acid,  sufficiently  to  produce  a  well-marked  calamine  tint  in  a 


24  PEACTICAL  HISTOLOGY 

very  dilute  solution.  Sections  should  be  left  for  24  hours  in 
the  stain.  Preparations  may  be  overstained  by  it,  and  de- 
colourised by  alcohol.  It  succeeds  best  with  sections  from 
sublimate  preparations, 

Microchemical  stain  for  iron  (Macallum).  The  sections  (of 
alcohol-hardened  tissues)  are  placed  for  a  short  time  in  an  aqueous 
solution  of  pure  haematoxylin  (1  to  300).  Any  free  iron  in  the 
tissue  is  at  once  stained  black  by  the  reagent.  To  show  the 
presence  of  iron  which  is  in  organic  combination  the  sections 
require  to  be  previously  treated  for  a  short  time  with  96  p.c. 
alcohol  containing  10  parts  hydrochloric  acid  per  cent. 

Microchemical  stain  for  phosphorus  (Lilienfeld  and  Monti).  The 
sections  (fresh  or  hardened  in  alcohol)  go  first  into  a  solution  of 
nitro-molybdate  of  ammonia  (a  few  minutes  to  several  hours) ; 
they  are  then  thoroughly  washed  and  placed  for  some  minutes  in 
pyrogallol  solution.  They  are  then  again  washed  and  transferred 
through  absolute  alcohol  and  xylol  into  balsam.  Parts  containing 
phosphorus  are  stained  brown. 

FLUIDS  USED  FOR  CLEARING  AND  MOUNTING  SPECIMENS  OF 
TISSUES  AND  ORGANS,  AND  FOR  FIXING  THE  COVER-GLASS  OP 
'wet'   SPECIMENS 

Grlycerine,  either  pure  or  diluted  with  an  equal  amount  of 
water  or  in  the  form  of  glycerine  jelly,  is  a  most  valuable 
medium  for  mounting  permanent  histological  specimens.  Its 
high,  but  not  too  high,  refractive  index  enables  it  to  render 
clear  many  of  the  details  of  an  object  which  might  from  the 
otherwise  too  great  opacity  of  the  tissue  remain  obscure.  At 
the  same  time  it  is  desirable  for  some  objects  that  this  clearing 
should  be  tempered  ;  a  result  easily  attained  by  diluting  the 
glycerine.  Dilution  is  also  necessary  in  the  case  of  specimens 
stained  with  aniline  dyes,  most  of  which  would  be  dissolved 
out  by  strong  glycerine.  In  these  instances,  where  diluted  gly- 
cerine is  employed  for  mounting,  it  becomes  necessary  to  pre- 
vent evaporation  of  the  water,  and  with  this  object  the  edges  of 
the  cover-glass  are  surrounded  or  '  ringed '  with  some  fatty  or 
resinous  material  .     For  temporary  purposes  a  film  of  olive  oil 


CLEARING  AND  MOUNTING  SPECIMENS 


26 


Pig.  14 


or  molten  paraffin  led  round  tlie  edge  of  the  cover-glass  is 
sufficient,  but  for  permanent  preparations  the  best  fixing  fluid 
for  the  cover-glass  is  gold  size,  which  is  painted  round  with  a 
small  camel-hair  pencil.  For  the  fixation  to  be  .successful  it 
is  necessary  that  the  surfaces  of  the  cover-glass  and  slide  to 
which  the  gold  size  is  applied  should  be  dry  and  clean,  and 
not  wetted  by  the  glycei'ine  used  in  mounting.  When 
glycerine  jelly  (which  must  be  warmed  for  use)  is  employed 
the  cover-glass  need  not  be  '  ringed.' 

Canada  balsam  dissolved  in  xylol  (xylol  balsam)  is  the 
other  chief  fluid  used  for  mounting, 
and  at  the  same  time  '  clearing,' 
preparations.  The  refractive  index 
of  this  substance  is  so  high  that  un- 
stained preparations  and  parts  of 
stained  preparations  which  have  re- 
mained relatively  little  stained  by 
the  dyes  used  are  rendered  perfectly 
clear  and  transparent,  and  the  de- 
tails of  structure  in  these  are 
almost  invisible,  thereby  showing 
up  by  contrast  the  details  of  the 
stained  portions  of  the  tissue.  Xylol 
balsam  is  chiefly  used  for  the 
mounting  of  stained  sections.  As  it 
is  absolutely  immiscible  with  water 
all  specimens  which  are  mounted  in 

it  must  first  be  completely  dehydrated.  This  is  effected  by 
immersing  them  in  absolute  alcohol.  Alcohol,  however,  is  also 
immiscible  with  balsam,  and  the  specimens  must  therefore  first 
be  passed  through  an  essential  oil  which  will  mix  with  either 
alcohol  or  balsam.  The  one  most  commonly  used  is  oil  of 
cloves,  and  this  has  the  advantage  that  the  alcohol  used  for 
dehydration  need  not  be  absolute,  for  the  oil  of  cloves  will  take 
up  a  little  water.  Other  essential  oils  which  are  employed  are 
oil  of  bergamot,  oil  of  cedar,  oil  of  turpentine,  and  xylol. 


Bottle  for  xylol  balsam 


26 


PKACTICAL  HISTOLOGY 


Bammar  varnish  is  sometimes  used  in  place  of  xylol 
balsam.  It  is  made  by  dissolving  dammar  resin  in  a  mixture 
of  equal  parts  of  warm  benzine  and  turpentine  ;  the  solution 
is  to  be  filtered  through  paper  wetted  with  chloroform. 

The  xylol  balsam  or  dammar  varnish  is  best  kept  in  a 
bottle  such  as  that  shown  in  fig.  14.  It  is  provided  with  a 
cap,  ground  to  fit  accurately  to  the  neck  of  the  bottle,  and 
with  a  loose  glass  rod  for  dipping  out  the  solution. 


METHODS   OP    PREPARING   SECTIONS 

After  the  preliminary  piocesses  of  fixing  and  hardening  are 
completed,  sections  can  be  prepared  from  a  tissue  or  organ.  It 

Fio.  15 


"T V" --"-p---p- 

I  I  11 

I  I  II 


Outline  showing  the  manner  in  which  a  small  piece  of  paper  is  to  be  folded  to 
make  an  embeddina'  trough 


is  now  usual  to  cut  them  with  the  aid  of  some  sort  of  microtome, 
but  for  certain  purposes  it  is  still  desirable  to  cut  by  hand 
with  a  razor,  which  is  usually  then  wetted  with  spirit.  The 
piece  of  tissue  to  be  cut  is,  if  large  enough,  held  in  the  left 
hand  ;  if  small  it  may  be  grasped  in  a  split  cork  or  in  a  piece 
of  alcohol  hardened  liver,  or  it  may  be  fixed  in  a  small  trough 
of  paper  or  lead  foil  (figs.    15,  16,    17)  and  molten  paraffin 


PIIEPAKATION   OF   .SECTIONS 


27 


poured  in  so  as  to  enclose  it  completely.     The  razor  must 
be  held  l>orizont;illy  and  Hooded  with  spirit  (fig.  18). 

For  cutting  sections  with  a  inicrotorac  it  is  desirable  to 
securely  fix  the  tissue  by  alhnving  it  to  he  pei-meated  with 
some  material  which  will  set  to  a  con.si.stence  suitable  for  slicing. 

Fi(i.  l(i 


Embedding  trough  inadi'  from  a  piece  of  juiijcr  of  the  size  shown  in  fig.  15,  with 
one  side,  6,  completed ;  the  other,  a,  only  half  finished,  so  as  to  show  the 
manner  in  which  the  corners  are  folded  and  fixed 


Fk;.  17 


Embedding  trough  of  lead  foil  placed  on  a  cork,  and  with  a  piece  of  tissue  in 
situ.  All  that  is  further  necessary  is  to  fill  the  trough  with  molten 
paraffin 

The  materials  which  are  commonly  used  for  this  purpose  are 
gum  (frozen),  collodion  or  celloidin,  and  paraffin,  and  the 
methods  are  termed  respectively  the  freezing,  the  celloidin  or 

collodion,  and  the  paraffin  methods. 


28 


PEACTICAL  HISTOLOGY 


Freezing  method. — This  is  by  far  the  most  expeditious 
method  of  preparing  sections,  and  for  many  histological  pur- 
poses is  as  good  as  any  other. 

A  thin  piece  of  the  hardened  tissue  is  first  soaked  in  water, 
then  placed  in  a  thin  syrup  of  gum  arabic  or  dextrine  for  a  few 
hours,  and  then  on  the  plate  of  a  freezing  microtome  (fig.  19). 
On  the  under  surface  of  this  plate  an  ether  spray  is  now 
allowed  to  play  until  the  tissue  is  just  frozen  through  ;   it  is 

Fict.  18 


Process  of  cutting  sections  of  an  embedded  tissue 


better  that  it  should  not  be  too  hard  frozen.  Sections  are  now 
made  with  the  plane-iron  or  razor  provided  with  the  microtome, 
the  freezing  plate  on  which  the  tissue  lies  being  gradually 
screwed  up  as  the  successive  sections  are  taken.  The  sections 
are  placed  in  water  to  dissolve  away  the  gum,  and  the  best  are 
then  to  be  selected  and  treated  with  such  staining  and  other 
reagents  as  may  be  desired.  They  can  be  readily  mounted  at 
once  in  glycerine,  or  they  may  be  passed,  after  staining, 
through  water,  rectified  spirit,  alcohol,  and  oil  of  cloves,  and 


PREPARATION  OF   SECTIONS 


29 


mounted  in  balsam  in  the  manner  desciil^ed  below   under  the 
paraffin  method. 

Collodion  method. —This  is  useful  for  preparing  sections 
of  large  objects  in  which  it  is  desirable  to  hold  the  several 
parts  securely  together  during  the  staining  and  subsequent 
treatment  of  the  sections.     The  organ  must,  after  hardening, 

Fui.  Ill 


microtome 


0,  object,  soaked  iu  gum  .aiid  placet!  on  metal  plate  of  object-bolder,  c  ;  b,  box  in  which 
the  ether  coDects  after  spraying  on  the  under  sm-face  of  the  plate  ;  c'.  extra 
holdei"  for  paraflau-embedded  tissues  ;  m,  micrometer  screw  for  raising  object ;  m, 
razor  fixed  in  a  clamp,  and  moving  horizontally  on  the  axis  ,s  x,  along  with  the 
pivotted  bar  a  ;  e,  screw  for  tight(!ning  clamp  of  razor ;  X-,  handle ;  G,  stand  of 
microtome  fixed  to  tabic  l)y  clamping  screw,  d  ;  /(.  lioldei-  for  ether-bottle 

be  cut  into  moderately  thin  slices,  and  these  are  placed  in 
absolute  alcohol  for  a  short  time,  and  then  in  a  mixture  of 
equal  parts  of  alcohol  and  ether.  From  this  they  are  trans- 
ferred to  collodion  made  by  dissolving  1  part  of  pyroxylin,  or 
phytoxylin,  or  celloidin  in  1.5  of  the  above  mixture,  i.e.  more 
rhan  double  the  strength  of  the  collodion  of  the  Pharmacopoeia. 


30 


PRACTICAL  HISTOLOGY 


In  this  they  are  left  in  a  well- stoppered  bottle  for  some  hours 
or  days,  according  to  their  thickness.  Each  is  then  placed 
upon  a  brass  holder  of  appropriate  size  and  shape  (fig.  20), 
and  the  collodion  is  allowed  to  set  by  evaporation  of  the  ether. 
This  will  occur  within  a  few  minutes,  and  the  whole  is  then 
immersed  either  in  chloroform  or  in  85  per  cent,  alcohol. 
In  this  the  collodion  becomes,  in  a  few  hours,  of  a  consistence 
suitable  for  the  preparation  of  sections,  which  must  be  made 
with  a  microtome  the  knife  of  which  is  horizontal  and  is  kept 
flooded  with  alcohol.  It  is  sometimes  preferred  to  soak  the 
collodion  block  in  cedar-wood  oil  (after  dehydrating  with  96  p.c. 

Pig.  20 


Brass  holders  for  collodion- soaked  preparations 

a,  for  small  pieces,  is  simply  a  piece  of  angle  brass  sawn  ofE  a  strip  ;  &,  for  large  pieces,  is 
the  same  with  a  brass  plate  soldered  to  it 

alcohol)  and  to  cut  with  the  knife  wetted  with  the  same  fluid. 
The  most  suitable  microtome  for  collodion-embedded  tissues 
is  the  inclined  plane  mic7-otome  (fig.  21)  :  the  knife  must  be 
set  as  obliquely  as  possible.  If  the  tissue  had  not  been  stained 
in  bulk  the  sections  may  now  be  stained,  the  methods  employed 
being  the  same  as  for  sections  cut  by  the  freezing  microtome. 
It  is  generally  desirable  to  keep  the  collodion  in  these 
sections,  so  that  they  may  be  better  held  together.  They  must 
therefore  not  be  placed  in  absolute  alcohol,  nor  in  oil  of  cloves, 
for  the  former  softens  and  the  latter  dissolves  collodion,  but, 
after  dehydrating  in  96  p.c.  alcohol,  oil  of  cedar-wood  may  be 


PREPARATION   OK   SECTIOMS 


HI 


used  for  clearing  tlicni,  ami  they  can  then  be  mounted  in 
xylol  balsam. 

Paraffin  method. — This  is  the  most  genfrally  useful,  and  for 
most  purposes  the  best  method  of  preparing  thin  sections  of  a 
tissue.  Moreover,  it  enables  any  number  of  successive  sections 
to  be  taken  and  kept  in  their  serial  older  ;  a  point  of  funda- 
mental importance  in  practical  embryology  and  morphology, 
and  of  some  importance  in  certain  branches  of  histology. 

The  tissues  to  be  embedded  must  always  be  ultimately 
placed,  whatever  fixing  and  hardening  fluids  have  been  used,  in 

Fi<;.  21 


Inclined  plane  microtome 

The  object,  o,  is  fixed  in  tlie  clamp  h,  which  is  attacheil  to  tlie  object  slider,  o  s,  slidingr 
up  an  inclined  plane,  the  exact  amount  of  movement  being  determined  by  the  micro- 
meter screw,  J.  Tlie  knife  is  tixeil  upon  another  slider  which  moves  horizontally  at 
the  back  of  the  instrument 

strong  alcohol.  It  is  advantageous  that  they  should  be  stained 
in  bulk,  as  this  saves  the  somewhat  tedious  process  of  staining 
the  individual  sections,  but  it  is  not  always  possible  to  effect 
bulk-staining  satisfactorily.  From  alcohol  the  tissue,  in  suf- 
ficiently thin  pieces,  is  placed  in  oil  of  cedar-wood  until 
soaked    through.'      To    facilitate  the    penetration  the  oil  of 

•  Chloroform,  turpentine,  and  xylol  nniy  be  used  instead  of  oil  of  cedar- 
wood  ;  they  must  be  preceded  by  absolute  alcohol.  For  delicate  objects  the 
transference  from  alcohol  to  paraffin  is  made  gradually  thi-ough  alcohal  ami 
chloroform,  pure  chloroform,  and  paraffiv  dissolved  in  chloroform,  the 
chloroform  being  finally  driven  ofT  by  gentle  heat;  but  this  long  i)rocess  is 
rarely  necessary  in  vertebrate  histology. 


32  PEACTICAL  HISTOLOaY 

cedar-wood  may  be  gently  warmed  (to  40°  C).  After  it  has 
completely  penetrated,  the  piece,  if  small,  is  transferred  to  a 
shallow  glass  vessel  [e.g.  a  watch-glass)  which  has  been  smeared 
with  glycerine,  and  into  which  paraffin  of  melting  point  45° 
to  50°  C.  has  been  poured,  and  kept  just  molten  in  a  suitably 
regulated  oven  or  water-bath.  In  this  it  is  left  for  half  an 
hour  or  several  hours,  according  to  size.  The  vessel  is  then 
removed  and  cooled  as  quickly  as  possible.  When  hard  set 
the  whole  is  turned  out  of  the  watch-glass,  and  the  excess 
of  paraffin  cut  away,  leaving  the  tissue  in  the  middle  of  a 
small  square  block  of  paraffin.  If  the  piece  is  larger,  it  is 
placed  for  some  hours  in  a  larger  vessel  of  melted  paraffin, 

Fig.  22 


Mould  for  paraffin 

and  this  is  then  poured  into  a  mould  made  of  paper  or  of 
lead  foil  (figs.  16,  17),  or  by  placing  together  two  L-shaped 
pieces  of  brass  (fig.  22),  the  tissue  being  arranged  with  warmed 
needles  within  the  paraffin,  or  fixed  in  place  beforehand  by  a 
pin  (fig..  17).  In  either  case  the  result  is  that  the  tissue 
which  has  been  soaked  with  paraffin  is  also  completely  sur- 
rounded and  supported  by  paraffin  of  the  same  consistence, 
and  is  thus  in  a  position  to  offer  a  uniform  resistance  to  the 
cutting  instrument. 

The  block  of  paraffin  is  now  fixed  in  the  desired  position 
in  the  holder  of  a  microtome.   Either  of  those  which  have  been 


MICROTOMES  ;-}3 

already  mentioned  for  freezing  or  fur  celloidin  will  serve  foi* 
cutting  paratlined  tissues,  but  the  knife  must  always  be  used 
dr\',  and  set  exactly  transversely.  The  three  instruments 
immediately  to  be  described  are,  however,  better  adapted  for 
the  purpose. 

The  tripod  microtome  (fig.  24). — Thi.s  consists  of  a  small 
frame  of  brass  or  cast,  iron  to  the  under  surface  oi*  one  end 
of  which  an  ordinary  razor  can  be  clamped.  Of  the  three  feet 
of  the  tripod,  two  are  fixed  and  one  is  provided  with  an  adjust- 
able screw.  The  microtome  frame  is  moved  by  the  hand  over  a 
flat  piece  of  glass  upon  which  the  paraffin  block  containing  the 
tissue  has  been  attached  by  aid  of  heat,  and  successive  sections 

Vtc.  21! 


Box  for  storing  specimeus  enclosed  in  paraflin  blocks 

are  cut  by  lowering  the  cutting  edge  of  the  razor  by  a  slight 
turn  of  the  screw  adjustment,  and  sliding  the  holder  over  the 
glass.  This  is  not  only  a  very  efficient,  but  also  a  very  cheap 
form  of  microtome,  for  the  frame  can  be  made  for  two 
or  three  shillings. ' 

If  it  is  not  required  to  keep  the  sections  in  series  they  may 
be  placed,  as  soon  as  they  are  cut,  in  turpentine  or  xylol,  which 
quickly  dissolves  tlie  paraffin.  Ef  the  tissue  has  been  stained 
in  bulk  all  that  is  now  necessary  is  to  mount  one  or  more 
sections  in  xylol  balsam.  But  if  the  sections  require  to  be 
stained  they  are  passed  first  from  xylol  into  absolute  alcohol, 

^  Jl.ide  by  A.  Kershaw,  Cankerwell  Lane,  Leeds. 

D 


34 


PEACTICAL   HISTOLOaY 


then  into  rectified  spirit,  then  into  distilled  water,  then  into 
the  stain,  e.g.  solution  of  hsemalum.  After  staining  they  are 
transferred  successively  to  water,  rectified  spirit,  absolute 
alcohol,  and  xylol,  and  are  at  length  ready  to  be  mounted  in 
xylol  balsam.  These  transferences  are  best  effected  in  glass 
or  earthenware  pots,  provided  with  well-fitting  covers  (fig.  25), 
and  the  sections  should  be  left  at  least  three  minutes  in  each 
fluid. 

Fig.  24 


Tripod  microtome  (Bircli's  pattern) 


The  preparation  of  serial  sections.  The  rocking  micro- 
tome.— In  this  instrument  (fig.  26)  an  ordinary  razor  is  also 
used,  but  it  is  fixed  vertically  with  the  edge  uppermost, 
and  the  tissue  is  moved  over  it,  the  parafiin  block  con- 
taining the  tissue  being  attached  to  a  lever  which  is  worked 
up  and  down,  and  also  gradually  thrust  forward,  as  the 
handle,  h,  is  worked.      In  this  way  successive  sections  are 


MICROTOMES 


36 


cut  automatically  of  uniform  lliickness.  Tf  the  sides  of 
the  paraffin  block  are  parallel,  and  the  paraffin  not  too 
hard,  the  successive  sections  adhere  to  one  another  edge  to 


Fin.  2.1 


edge  into  a  ribbon,  which  may  hold  together  for  a  considerable 
length.  Such  paraffin  ribbons  may  be  cut  by  other  microtomes, 
using  the  knife  dry,  and  witli  the  edge  at  right  angles  to  the 


Fu;.  2(; 


Rocki)ig'  microtome  (Cambridge  pattern) 

direction  of  movement,  but  instruments  like  the  locking  micro- 
tome, which  have  the  razor  fixed  vertically,  are  the  most  con- 
venient forms  for  this  purpose. 

D'i 


36 


PEACTICAL  HISTOLOGY 


Minot's  microtome. — In  this  instrument  the  razor  is  also 
j&xed  vertically,  and  the  block  of  paraffin  containing  the  em- 
bedded tissue  is  caused  to  move  vertically  up  and  down  over 
the  edge  of  the  razor,  with  an  alternate  movement  of  advance, 
which  is  capable  of  being  regulated  by  a  screw  adjustment  to 
the  utmost  degree  of  nicety.^ 

Methods  of  fixing  paraffin-cut  sections  in  series  upon  a 
slide. — The  following  methods  are  used  for  fixing  the  series 

Fig.  27 


Minot's  microtome 


or  ribbon  of  paraffin-cut  sections  upon  a  slide,  and  thus  keep- 
ing the  sections  in  order  whilst  being  treated  for  permanent 
mounting  : — A  ribbon  of  sections  having  been  cut  of  a  length 
suitable  for  mounting,  it  is  arranged    appropriately  upon  a 


1  Both  the  Cambridge  Scientific  Instrument  Co.  and  Messrs.  Bausch  &: 
Lomb  (for  Prof.  Minot)  have  recently  brought  out  new  microtomes,  as  im- 
provements upon  those  here  described  ;  but  thej"  are  larger  and?mueh  more 
expensive,  and  the  above  will  be  found  tr)  answer  every  practical  puriDose. 


FIXING   SECTIONS   '1^0   SLIKE  37 

carefully  cleaned  slide  which  is  fi-eely  wetted  with  water. 
The  slide  is  then  gently  warmed  until  the  sections  are 
flattened  out,  when  the  excess  of  water  is  drained  oft", 
and  the  slide  allowed  to  stand  in  a  warm  place  until 
the  water  has  completely  evaporated,  leaving  the  paraffin 
ribbon  adhering  to  the  glass.  The  slide  is  now  heated  until 
the  paraffin  just  melts  ;  it  is  then  allowed  to  cool  and  is 
immersed  in  xylol.  This  dissolves  the  paraffin,  but  the  sections 
are  left  sticking  to  tlie  slide.  If  the  tissue  was  stained  in 
bulk,  and  tlie  sections  are  therefore  already  sufficiently 
stained,  they  can  [be  at  once  mounted  in  xylol  balsam  and 
covered. 

Varioiis  solutions,  such  as  collodion  dissolved  in  oil  of  cloves 
and  slieUac  dissolved  in  creosote,  are  employed  with  a  view  to 
fixing  the  sections  more  securely.  They  are  chieflj'  necessary  for 
chromic-hardened  tissues.  In  any  case  it  is  well  to  combine  them 
with  the  water  method,  for  it  is  only  by  the  latter  that  a  series 
of  paraffin  sections  can  be  properly  ffattened  out.  They  should  be 
brushed  over  the  sections  when  they  are  completely  dry,  and  before 
the  parafhn  has  been  dissolved  by  xylol.  Diluted  white  of  egg  is 
also  used  vevy  freqiiently  for  the  same  purpose.  A  solution  which 
can  be  kept  for  some  time  is  made  by  shaking  up  50  c.c.  egg-white 
with  50  c.c.  glycerine  and  dissolving  1  gramme  salicylate  of  soda 
in  the  mixtiu-e.  The  slide  which  is  to  receive  the  sections  is 
smeared  thinlj'  over  with  this,  then  covered  with  water,  and  the 
series  of  sections  is  laid  in  position.  It  is  now  gently  warmed 
imtil  the  paraffin  is  softened  and  the  sections  lie  flat,  when  the 
excess  of  water  is  removed  by  draining  and  evaporation.  Tlie  slide 
is  then  immersed  for  an  liour  in  a  mixture  of  alcohol  and  etlier  to 
coagulate  the  albiunen,  then  in  xylol  to  dissolve  away  the  paraffin, 
and  finally  tlie  sections,  if  already'  stained,  may  be  covered  in 
balsam. 

But  if  not  stained  in  bulk  a  further  procedure  is  neces- 
sary. The  slide,  after  immersion  in  xylol,  is  placed  in  absolute 
alcohol  for  a  few  minutes,  then  in  50  per  cent,  spirit,  then  in 
the  staining  fluid,  e.tj.  luemaluni.  After  the  sections  are 
sufficiently  stained  the  slide  is  transferred  successively  to 
water,  50   per  cent,  spirit,  absolute   alcohol   or   methylated 


38  PEACTICAL  HISTOLOGY 

spirit,  and  xylol  or  oil  of  bergamot  or  oil  of  cloves,  ^  and  finally 
the  sections  are  covered  with  xylol  balsam  and  mounted. 

Special  oblong  porcelain  vessels  with  grooved  ends  are  made 
for  holding  the  series  of  fluids  required  for  effecting  these 
transferences  (fig.  28)  ;  they  should  be  provided  with  well- 
fitting  covers.  The  slides  are  placed  bodily  into  them.  The 
series  of  operations  may  be  performed,  if  desired,  by  pouring 
the  successive  fluids  freely  over  the  sections.  They  may 
also  be  performed  with  the  sections  fixed  beforehand  to  a 
cover-glass  instead  of  upon  a  slide.  The  following  diagram 
may  serve  to  show  at  a  glance  the  several  transferences 
which  are  necessary  in  order  to  stain  and  permanently 
mount  parafiin-cut  sections  : — 

DiAGBAM   TO    SHOW   METHOD    OP    TbEATMENT    OF   PaKAPFIN-CUT    SECTIONS 

1.  Place  on  a  slide  in  water  :  warm  gently. 

I 
2.  Drain  off  water  :  allow  to  dry  completely. 

3.  Warm  until  paraffin  is  just  melted. 

I 
4.  Xylol. 

If  tissue  ia  already  stained  in  bulk.       |         If  sections  require  to  be  stained. 

I  i 

Xylol-balsam  5.  Absolute  alcohol. 

I 
6.  50  per  cent,  alcohol. 

For  sections  cut  by  the  freezing  or  by  7.  Stain  [e.g.  hsemalura). 

the  celloidin  methods,  processes  1  to  6  of  | 

the  accompanying  table  are  omitted,  if  the  8.  Water, 

tissue  has  not  been  stained  in  bulk.     If,  | 

however,  it  has  been  already  stained  in  9.  50  per  cent,  alcohol, 

bulk,  the  sections  need  only  be  piit  through  | 

the  last  three  of  the  series.  10.  Absolute  alcohol  or 

methylated  spirit. 

11.  Xylol  or  bergamot  oil 
or  clove  oil. 

Xylol-balsam. 

^  Xylol  needs  to  be  preceded  always  by  absolute  alcohol,  but  as  this  is 
somewhat  expensive  methylated  spirit  may  be  used  instead,  in  which  case 
oil  of  cloves  or  oil  of  bergamot  must  be  used  as  the  intermediary  between 
alcohol  and  Canada  balsam. 


GENERAL   DIRECTIONS 


39 


GENERAL     DIRECTIONS     FOR     WORK.       METHODS     OF     URAWINO, 
PUOTOORAPIIINC;,    AND    MEASURING    MICROSCOPIC    OBJECTS 

IJi'forecoimiieiiciiig  see  that  the  table  is  in  order  and  clean, 
and  that  everything  is  at  hand  that  is  usually  wanted  for 
histological  work.  Especially  look  carefully  to  the  glasses  of 
the  microscope  that  there  is  no  dust  or  other  impurity  on 
them.     If  any  glycerine  or  Canada  balsam  should  have  found 

Fui.  2H 


its  way  on  to  the  objective,  as  is  often  the  case  when 
sufficient  care  is  not  taken  in  placing  a  preparation  upon  or 
removing  it  from  the  stage,  they  are  to  be  rubbed  off,  the 
former  by  a  cloth  wetted  with  water,  the  latter  with  a  little 
spirit.  Before  beginning  to  jDrepare  a  tissue  it  will  be  necessary 
to  look  over  the  description  of  the  mode  of  making  each  pre- 
paration in  order  to  knoAy  what  vessels  and  what  reagents  to 
get  together.  Otherwise  many  a  specimen  will  be  spoiled  by 
being  left  too  long  in  one  Huid,  whilst  the  one  to  which  it 
should  be  transferred  is  being  got  ready.  The  cover-glass 
should  always  be  cleaned  and  dried  before  commencing,  and 
placed  I'eady  to  hand  in  some  situation  where  it  is  not  likely 
to  get  broken.     It  is  well  always  to  put  the  cleaned  cover- 


40 


PEACTICAL  HISTOLOGY 


glasses  in  the  same  place — say  on  the  foot  of  the  microscope 
or  in  special  clips  (fig.  8) — otherwise  when  wanted  quickly  it 
is  often  difiicult  to  find  them. 

Every  specimen  that  is  to  be  kept  must  be  distinctively 
labelled  as  soon  as  made  ;  and  if  there  is  anything  of  import- 
ance to  remember  about  it  this  must  be  at  once  entered  in 
the  notebook,  which  no  one  who  is  working  with  the  micro- 
Scope  should  be  without. 

Modes  of  drawing  microscopic  ohjects. — The  student 
should   never  trust   to  the  transient   impression  of   form  or 

Fig.  29 


Camera  lucida,  for  tracing  the  outlines  of  an  object  without  tilting  or  otherwise 
disturbing  the  microscope.     The  metal  ring  fits  on  the  upper  end  of  the 
microscope  tube,  and  the  aperture,  a,  is  placed  immediately  over  the  eye- 
glass, this  part  of  the  camera  being  somewhat  more  depressed  than  is  repre- 
,  sented  in  the  figure 

structure  which  the  mere  glance  at  a  microscopic  preparation 
conveys,  but  should  always,  whether  naturally  a  good  draughts- 
man or  not,  endeavour  to  perpetuate  the  impression  so  obtained 
by  a  careful  sketch  showing  the  more  important  points  which 
the  preparation  illustrates.  Even  without  skill  a  little 
practice  soon  enables  a  sketch  to  be  produced  which  gives  a 
fairly  good  idea  of  the  appearances  seen,  and,  however  rough 
it  may  be,  serves  materially  to  assist  the  memory. 

But  if  greater  accuracy  of  delineation  is  desired  the  out- 
lines may  be  traced  with  a  camera  lucida.     The  simplest  for 


.MODKLLINO 


41 


ordinary  use  is  thiit  of  Abbe,  Ijut  the  principle  of  all  is  the 
same,  viz.  to  throw  together  upon  the  retina  an  image  of  the 
object  under  examination  and  of  the  paper  on  which  the 
drawing  is  to  be  made.  The  image  of  the  object  then  appears 
projected  upon  the  paper,  and  its  outlines  may  be  followed  with 
the  pencil.  In  Abbe  and  Zeiss's  instruments  the  surface  of 
the  paper  is  reflected  by  mirrors  to  the  eye  which  is  applied 
to  the  microscope,  the  object  being  viewed  directly  ;  in  some 
other  forms  the  paper  is  viewed  directly  and  the  image  of  the 
object  by  reflection.     In  using  the  camera  neither  tlie  paper 

Frc.  :50 


C'umer.i  lucida  of  Aldje 

The  eye  lookiui.'  ilowii  the  microscoiic  is  placed  at  o,  aurl  the  image  of  the  paper  is 
reflected  by  tlie  mirror  and  prisms  along  the  lines  s^,  sp,  w,  o,  to  the  eye 

nor  the  object  should  be  too  brightly  illuminated,  and  the 
paper  should  not  ]je  placed  on  the  table,  but  on  a  box  or  block 
at  about  the  level  of  the  stage  of  the  microscope.  It  is  not 
possible  to  trace  the  finer  details  with  the  camera,  but  only  the 
broader  outlines  of  objects. 

Modelling. —  The  modelling  of  microscopic  objects  is  required 
more  in  morphological  than  in  histological  studies,  but  a  word  here 
as  to  the  method  may,  nevertheless,  not  be  inappropriate.  The 
object  is  to  obtain  a  model  of  an  organ  reconstructed  from  serial 
sections.  The  object  is  attained  by  tracing  upon  wax  plates  of 
uniform  thickness  the  oxitlines  of  the  organ  as  they  appear  in 
sections  taken  at  successive  levels ;  the  wax  plates  then  have  the 
superfluous  parts  cut  away,  and  when  piled  upon  one  another  in 
series  a  majmified  model  of  the  or^an  is  obtained. 


42 


PEACTICAL  HISTOLOGY 


Fig.  si 


Micro-photographic  apparatus.     One-seventh  the  natural  size 


MICRO-IMIOTOdlv'AI'HV 


43 


J^t>  B  o 

a  a  e;.^ 

a  «)  o  a 

3  §3.0 

oi  2« 

_  8  t»  = 

SiH=   "     . 

1^^;^ 

a   m  D  =  r'5 

^«S-2o 

•=5^2s 

5=   tjl]~   '.y  •- 

==•1=^- 

:/> 

S 

"-^  7.  :3-^ 

■+-» 

'f-  c3  =  0 

3-ii^ 

p< 

odSSS 

^§•3  5  s 

p  K  0  s  > 

FTo 

^3  -  "i  ^ 

o 

osas-a 

o 

->^  15  =  2  so 

& 

l!1ll 

o 

c: 

o'o^  c  jf 

0  0  f-1  ^  — 

w 


o  P«  s  ^  ^ 


^  s;  o  ::.■-  ^ 


44  PEACTICAL  HISTOLOGY- 

In  order  to  maintain  the  proper  relative  position  of  the  outUnes 
one  above  the  other  the  paraffin  block  must  be  marked  with  lines 
at  right  angles  to  the  planes  of  section,  and  this  can  be  done  by 
cutting  clefts  longitudinally  in  it,  along  one  or  more  sides,  and 
filling  them  with  lampblack  or  vermilion.  The  sections  of  these 
marks  are  also  sketched  in  upon  the  wax  plates,  and  indicate 
exactly  how  the  plates  are  to  be  superposed.  The  plates  can  be 
stuck  together  by  warming  their  edges.^ 

Micro-photography. — Certain  microscopic  objects  lend  themselves 
better  than  others  to  photographic  reproduction,  but  it  is  in  any 
case  only  the  thinnest  and  best  specimens  which  yield  results  at  all 
•commensurate  with  the  time  and  pains  it  is  usually  necessary  to 
expend  to  obtain  really  good  results  in  micro -photography.  The 
method  employed  needs  no  special  description.  An  ordinary 
camera  may  be  used,  the  lens  being  removed,  and  in  place  of  it  the 
upper  end  of  the  microscope  tube — which  must  be  placed  horizon- 
tally for  this  purpose — inserted,  movably  but  light-tight.  But  it 
is  better  to  have  a  special  camera  made  with  a  body  at  least  twice 
as  long  as  usual.  It  may  be  arranged  either  vertically  or  horizon- 
tally (figs.  31  and  32).  Apochromatic  objectives  should  be  em- 
ployed if  possible,  and  they  may  be  iised  either  withoiit  eye-piece 
or  with  a  projection  eye-piece,  whereby  it  is  easier  to  obtain  a  flat 
field,  although  with  considerable  loss  of  light.  The  best  source  of 
light  is  either  an  arc  or  an  oxyhydrogen  light,  the  substage  con- 
denser being  used  for  high  powers.  For  low  powers  the  light  may 
advantageously  be  used  without  a  condenser.  In  all  cases,  and 
certainly  when  a  condenser  is  employed,  it  is  necessary  to  employ 
an  alum-  or  a  water-cell  to  cut  off  the  heat  rays.  The  best  dry 
plates  for  micro-photography  are  the  isochromatic,  preferably  with 
antihalation  backing. 

]\Iicro-photographs  can  rarely  take  the  place  of  drawings,  never 
certainly  for  the  student.  But  they  are  often  useful  for  assisting 
the  draughtsman,  and  for  this  purpose  no  form  of  print  is  so  con- 
venient as  those  upon  ferro-prussiate  paper.  These  are  printed  in 
sunlight  and  developed,  and  fixed  simply  by  washing  with  water. 
When  dry,  the  print,  including  just  so  much  detail  as  is  desired, 

1  Full  details  of  the  method  of  modelling  from  sections  will  be  found  in 
G.  Born,  '  Die  Plattenmodellirmethode,'  Arch.  f.  viikr.  Anat.  xxii.  p.  584 ; 
H.  Kastschenko,  'Methode  zur  genauen  Eeconstr.  vls.vj.,'  Arch.  f.  Anat.  1886, 
p.  388 ;  and  H.  Strasser,  '  Ueber  die  Methoden  der  plast.  Reconstruction,' 
Zeitschr.  f.  wiss.  Mikr.  iv.  p.  168.  Also  in  a  later  paper  by  Born  in  Zeitschr. 
f.  wiss.  Mikr.  v.  p.  433. 


MEASURINC 


15 


may  be  traced  over  with  u  lead  pencil,  aad  the  blue  colour  then 
removed  by  immersion  in  carbonate  of  soda  solution,  followed  first 
by  dihite  (1  per  cent.)  hydrochloric  acid  and  then  by  water.  This 
method  of  delincatin}?  microscopic  objects  will,  in  many  cases,  be 
found  extremely  practicMl. 

Methods  of  measuring  objects  under  the  microscope. — If  while  tliu 
one  eye  looks  do\\  n  the  tube  tlic  other  is  allowed  to  remain  open, 
an  image  of  the  object  will  appear  projected  on  the  table  at  the  side 
of  the  microscope,  and  it  is  not  difticult  to  mark  off,  upon  a  sheet 

Fio.  33 


Ocular  micrometer,  natural  size 
Part  of  the  side  is  represented  as  broken  away  to  sliow  the  fielil-glass  at  the  bottom,  and 
the  micrometer-glass,  m,  a  little  below  tlio  middle.    The  collar,  r,  serres  to  vary  the 
distance  oi  the  eye-glass  from  the  micrometer 

of  paper  placed  there,  the  pomts  between  which  the  measurements 
are  to  be  taken.  The  preparation  is  then  removed,  and  a  stage 
micrometer  is  substituted  for  it,  the  parts  of  the  microscope  being 
left  in  the  same  condition  as  before.  The  staf/c  viicronieter  is  a 
glass  slide  on  which  tine  equidistant  parallel  lines  have  been  ruled 
with  a  diamond.  They  are  usuall\-  ruled  transversely  to  the  long  axis 
of  the  slide,  but  it  is  better  to  have  them  parallel  to  the  long  axis. 
The  distance  between  the  lines  is  marked  on  the  sUde ;  it  is  gene- 
rally either  the  xib^h  and  rrfffTrth  part  of  an  inch,  or  the  j^th  and 
T^^th  part  of  a  millimetre.     The  lines  are  observed  with  the  micro- 


46 


PEACTICAL  HISTOLOGY 


scope  in  the  same  way  as  the  object,  and  their  image  can  of  course 
be  similarly  projected  upon  a  sheet  of  paper  and  there  marked 
down.  The  distances  between  the  lines  being  known,  it  is  easy,; 
by  comparison  of  the  two  markings,  to  find  out  the  distance  between 
the  opposite  points  of  the  object.  The  projection  of  the  lines  of  the 
stage  micrometer  can  be  still  more  easily  effected  with  the  aid  of  a 
camera  lucida. 

Fig.  34 


Lines  of  stage  micrometer  viewed  with  an  ocular  micrometer 

The  finer  lines  are  those  of  the  stage  miorometer  ;  about  eighteen  of  the  lines  of  the  ocular 
micrometer  are  comprised  in  one  of  the  larger  intervals  between  them,  so  that,  if  these  ^ 
intervals  represent  ^^  inch,  the  subdivisions  of  the  ocular  micrometer  will  represent 
TT3u  iiwh 

The  microscope  may  be  provided  with  an  eye-piece  micrometer 
(fig.  33).  This  is  an  ordinary  ocular  with  a  flat  piece  of  glass  (m), 
having  a  scale  ruled  upon  it  by  a  diamond,  inserted  between  the 
field-glass  and  eye-glass.  The  value  of  the  divisions  of  the  scale 
should  be  determined  once  for  all  for  each  objective  by  observation 
of  a  stage  micrometer  (see  fig.  34),  the  tube  of  the  microscope  being 


MArrNIFYING  POWER  OF  MICROSCOPE  47 

fally  drawn  out,  aiul  sliould  bo  markeil  on  the  ocular  ;  in  subse- 
quently nsiiifr  it  for  measurement  all  that  is  necessary  is  toseo  how 
many  divisions  of  the  scale  the  object  under  examination  covers. 
Thus,  supposing  it  had  been  found  by  examination  of  a  stage  micro- 
meter that  with  the  high-power  objective  and  the  tube  drawn  out  each 
division  of  the  eye-piece  micrometer  was  worth  TgVjj  inch,  any  object 
which  when  viewed  by  the  same  objective  and  length  of  tube  took 
up  three  divisions  of  the  eye-piece  micrometer  would  measure 
fg^Vtrths  or  nliTith  of  an  inch. 

The  advantage  of  the  eye-piece  micrometer  is  that  when  its 
values  are  once  ascertained  the  size  of  an  object  can  be  read  off  at 
once. 

Determination  of  the  magnifying  power  of  a  microscope. — The 
magnifving  power  of  a  microscope  is  determined  by  comparing  the 
distance  between  the  lines  of  the  stage  micrometer,  as  they  appear 
imaged  upon  the  paper,  when  this  is  exactly  ten  inches  '  from  the 
eye,  with  the  known  interval  between  them.  For  instance,  if,  with 
the  high-power  objective  and  the  ordinary  ocular,  the  interval  of 
yj^firfth  of  an  inch  of  the  micrometer  was  rejjresented  on  the  paper 
by  a  space  of  half  an  inch,  this  interval  is  magnified  as  many 
times  as  the  x^iVTrtl^  of  an  inch  will  go  into  half  an  inch,  that  is  to 
say  500  times;  and  every  other  object  under  similar  conditions  is 
magnified  to  a  lilce  extent. 

The  enlargement  thus  obtained  may  be  determined  once  for  all 
for  each  objective,  the  same  ocular  being  used  and  the  tube  being 
drawn  out  to  the  same  extent— say  to  the  full  length — in  each  case, 
and  scales  may  be  made  representing  the  intervals  between  the 
micrometer  lines  under  the  different  powers.  For  pxu'poses  of 
measurement  it  will  then  only  be  necessary  to  compare  the  projected 
image  of  an  object  with  the  scale  which  was  made  under  like  con- 
ditions, without  again  making  use  of  the  stage  micrometer. 

'  This  distance  is  taken  arbitrarily. 


48  PEACTICAL  HISTOLOaY 


CHAPTER   I 

THE    BLOOD 

Examination  of  human  blood. — A  drop  of  blood  may  be 
most  conveniently  obtained  for  examination  from  the  finger. 
It  is  generally  sufficient  to  give  the  end  of  the  forefinger  of 
the  left  hand  a  smart  prick  with  a  clean  needle,  in  the  thin 
part  of  the  skin  adjoining  the  root  of  the  nail,  squeezing 
firmly  with  the  right  hand  above  the  point  pricked  to  cause 
a  drop  to  exude.  If  necessary,  the  finger  may  first  be  con- 
gested by  tying  a  piece  of  string  tightly  round  it.  As  soon 
as  a  small  drop  of  blood  has  been  pressed  out,  take  up  a 
pre\'iously  cleaned  cover-glass  by  one  edge  with  forceps,  let 
the  drop  come  in  contact  with  the  lower  surface  of  the  glass 
near  the  opposite  edge,  so  that  a  little  adheres  ;  and  then, 
letting  this  edge  come  first  in  contact  with  the  upper  surface 
of  the  slide  near  its  middle,  gradually  lower  the  other  edge, 
which  is  still  held  in  the  forceps,  on  to  the  slide  (fig.  6,  p.  8). 
When  the  lower  blade  of  the  forceps  nearly  touches  the  slide, 
withdraw  the  instrument  carefully,  so  that  the  cover-glass 
may  now  rest  evenly  upon  the  slide  by  its  whole  under 
surface,  with  the  blood  in  a  uniform  thin  layer  between.  It 
is  important  not  to  let  the  cover-glass  down  too  suddenly,  for 
if  dropped  carelessly  on  the  slide  many  of  the  corpuscles  will 
be  broken  and  destroyed.  When  the  cover-glass  is  in  its  place, 
there  ought  to  be  just  enough  blood  entirely  to  fill  the  space 
between  the  two  glasses  ;  but  it  is  better  to  have  too  little  than 
too  much.  It  might  be  supposed  that  the  delicate  corpuscles 
would  be  crushed  between  the  cover-glass  and  slide,  but  they 


THE   BLOOD  49 

are  for  the  most  part  protected  frorn  this  by  tho  buoying-up 
of  the  cover-glass  by  the  liquid  in  which  they  float.  With  a 
little  practice  the  cover-glass  can  1)6  placed  in  position  equally 
well  witliout  the  use  of  forceps  by  holding  its  opposite  edges 
between  the  finger  and  thumb. 

The  preparation  made,  it  is  to  be  at  once  transferred  to 
the  stage  of  the  microscope,  and  examined  with  a  power  of 
from  300  to  r)00  diameters.  The  field  of  the  microscope  will 
be  seen  crowded  with  corpuscles  floating  in  a  clear  liquid. 
Probably  the  first  thing  which  will  strike  the  beginner  is 
the  very  faint  colour  which  the  so-called  red  corpuscles  pre- 
sent, and  probably  these  will  also  be  the  only  kind  of  cor- 
puscle that  he  will  at  first  be  able  to  distinguish.  But  if  at 
the  moment  of  observation  there  happens  to  be  a  current 
in  the  fluid — produced  either  accidentally  by  a  shaking  of  the 
room  or  a  draught  of  air,  or  purposely  by  gently  touching  the 
cover-glass  with  a  bristle — it  will  be  seen  that  while  most 
of  the  corpuscles  are  carried  along  by  the  current  two  or 
three  remain  sticking  to  the  glass,  whilst  the  otliers  are 
carried  past  them  ;  and  on  close  examination  it  will  further 
be  clear  that  these  are  of  a  different  nature  from  the  rest, 
being  entii-ely  devoid  of  colour  and  of  a  pale,  granular  appear- 
ance. They  are,  in  fact,  white  corpuscles,  and  once  seen  will 
be  easily  recognised  again,  even  Avhen  the  fluid  is  at  a  stand- 
still. 

Another  thing  that  will  be  made  manifest  by  any  motion 
in  the  fluid  is  the  biconcave  discoid  shape  of  the  red  corpuscles, 
for  as  they  roll  over  it  will  be  seen  that  their  outline  is  no 
longer  circular  as  when  lying  flat,  but  that  wlien  a  lateral  view^ 
of  the  discs  is  obtained  the  flattening  and  incurvation  of  the 
surfaces  become  evident. 

When  the  motion  in  the  layer  of  blood,  in  whatever  way 
it  may  have  been  produced,  is  subsiding,  it  will  be  seen  that 
whenever  one  corpuscle  comes  in  contact  with  another  the 
two  seem  to  be  in  some  way  attracted  to  one  another,  so  as  to 
adhere  closely  by  their  opposed  surfaces  ;  and  other  corpuscles 

E 


50  PEACTICAL  HISTOLOGY 

coming  in  the  same  way  in  contact  with  these  and  adhering, 
little  piles,  or  rouleaux,  are  thus  produced,  which  form  by 
their  junction  with  one  another  a  network,  extending 
throughout  almost  the  whole  of  the  preparation.  In  the  cords 
of  this  netwoik  nearly  all  the  red  corpuscles  are  involved, 
and  now  for  the  most  part  are  seen  edgeways  ;  but  in  other 
parts  of  the  preparation  where  the  layer  of  blood  is  very  thin 
■ — the  space  being  too  small  to  allow  the  corpuscles  to  stand 
edge  up,  and  to  combine  so  completely  to  form  rouleaux — they 
may  be  found  still  lying  flat  and  distinct  from  one  another  ; 
and  these  more  isolated  corpuscles  may  now  be  subjected  to 
careful  examination.  Keeping  a  single  red  corpuscle  in  view, 
if  it  be  brought  exactly  into  focus — that  is  to  say,  if  the 
microscope  be  so  adjusted  that  the  contour  of  the  corpuscle 
is  as  distinct  as  possible — it  will  be  observed,  with  the  power 
(^  inch)  which  is  at  present  being  employed,  that  the  middle 
part  appears  slightly  darker  than  the  rim,  whereas  if,  by 
means  of  the  fine  adjustment,  the  objective  be  now  brought 
somewhat  nearer  (lower),  the  middle  part  will  come  to  appear 
lighter. 

The  cause  of  this  is  probably  to  be  found  in  the  shajye  of  the  cor- 
puscle, the  middle  i^art  of  which  acts  upon  the  light  like  a  biconcave 
lens,  refracting  the  rays  of  light  which  are  transmitted  through 
it  slightly  outwards ;  so  that  if  the  objective  is  at  a  certain  distance, 
all  of  the  rays  which  traverse  the  central  part  do  not  reach  it, 
some  of  them  being  deflected  too  much  to  the  side  to  impinge  upon 
the  lower  glass  of  the  objective.  The  part  in  question,  therefore, 
looks  a  little  dimmer  than  the  somewhat  convex  marginal  part, 
whereas  when  the  objective  is  brought  nearer  all  the  rays  are  in- 
tercepted by  it,  and  the  middle  part,  owing  to  its  greater  thinness, 
appears  lighter  than  the  rim.  If  an  objective  of  very  short  focal 
length  is  used,  the  middle  part  of  the  corpuscle  will  be  the  lighter, 
even  when  the  focus  is  rightly  adjusted,  for  such  an  objective 
approaches  near  enough  to  intercept  the  outwardly  refracted  rays. 

With  the  exception  of  these  differences  of  shading  (which 
are  merely  dependent  upon  the  shape  of  the  corpuscle),  the 
red  corpuscles  present  a  perfectly  homogeneous  appearance. 


THE   BLOOD  51 

and  exhibit  in  tlie  fresh  condition  no  tendency  to  separate 
into  the  two  parts  of  which,  as  the  study  of  the  action  of 
reagents  will  show,  they  in  reality  consist.  But  there  may 
generally  be  noticed,  even  in  a  preparation  which  has  been 
made  with  the  greatest  care,  a  red  corpuscle  here  and  there 
Avhich  varies  from  the  prevailing  form,  having  become  more 
globular,  and  at  the  same  time  rather  smaller  in  diameter. 

These  retain  a  smooth  contour,  wliilst  others,  especially 
those  near  the  edge  of  the  preparation,  have  a  jagged  or 
crenate  margin,  as  if  set  with  little  projections,  and  such  pro- 
jections may  also  be  seen  on  the  surfaces  of  the  corpuscles  by 
carefully  adjusting  the  microscope.  This  change  of  form, 
which  is  very  charactei-istic  of  the  mammalian  red  corpuscles 
on  exposure,  seems  to  be  generally  caused  by  a  shrinkintr  of 
the  corpuscles,  induced  by  an  increase  in  the  density  of  the 
plasma  in  which  they  float  :  it  may  always  be  produced  by 
adding  salt  to  blood. 

Turning  now  our  attention  to  the  white  corpuscles,  not 
more  than  two  or  three  of  which  are  to  be  seen  in  each  field 
of  the  microscope  when  the  ordinary  high  power  is  beino- 
used,  and  which,  as  before  stated,  are  readily  distinguishable 
from  the  red  corpuscles  by  their  want  of  colour  and  their 
pale,  granular  aspect,  we  usually  notice,  if  seen  soon  after  the 
blood  is  drawn,  and  provided  they  are  not  pressed  down  by 
the  cover-glass,  that  they  are  spheroidal  and  completely 
motionless,  exhibiting  no  indications  of  vitality.  Some  of 
them  may  be  noticed  to  contain  a  small  group  of  well-marked 
granules,  much  coarser  than  the  excessively  fine  granules 
which  pervade  the  whole  substance  ;  and  in  conformity  with 
this  it  is  usual  to  describe  two  kinds  of  white  corpuscles — the 
finely  granular  and  the  coarsely  granular.  In  addition  to 
these  there  are  others  which  appear  almost  perfectly  clear  and 
hyaline.  As  a  rule,  before  the  addition  of  reagents,  no 
nucleus  is  visible  in  either  variety,  although,  as  will  be  after- 
wards seen,  one  or  more  is  always  present  in  each  ;  the  nuclei 
are  delicate,  however,  and  readily  obscured  by  the  granules 

e2 


52  PRACTICAL  HISTOLOaY 

of  the  protoplasm.  If  the  room  is  tolerably  warm  it  may 
happen  that  the  white  corpuscles  no  longer  preserve  their 
rounded  outline,  but  that  from  one  side  or  another  of  a  cor- 
puscle a  bud-like  process  extends  itself,  to  be  again  retracted 
into  the  body  of  the  corpuscle,  spontaneous  changes  of  form 
being  thus  effected  which  resemble  those  which  are  presented 
by  the  common  fresh-water  amoeba,  and  are  hence  termed 
*  amoeboid.'  But  in  a  cold  preparation  of  human  blood,  like 
that  under  examination,  these  movements  are  seldom  exten- 
sive, and  do  not  serve  to  eflfect  an  actual  change  of  place  in 
the  corpuscles  such  as  we  shall  see  to  be  the  case  in  a  prepara- 
tion which  is  artificially  warmed. 

Further,  there  may  generally  be  seen  in  a  preparation  of 
blood,  in  the  clear  interstices  between  the  rouleaux  of  red 
corpuscles,  a  number  of  minute  pale  granules  of  a  discoid 
shape  (hlood-platelets),  which,  if  present  in  quantity,  may 
be  closely  grouped  together  here  and  there  into  masses  of 
various  shapes  and  sizes,  which  the  beginner  is  sometimes 
apt  to  mistake  for  white  blood-corpuscles.  But  the  objects  in 
question  have  a  much  fainter  aspect,  and  nothing  resembling  in 
nature  the  amoeboid  movements  of  the  white  blood-corpuscles 
is  ever  observed  in  them. 

The  masses  in  question  are  often  of  considerable  size,  many 
times  larger  than  a  pale  blood-corpuscle.  As  Osier  has  shown, 
the  particles  which  compose  them  are  free  in  the  circulating 
blood,  and  only  run  together  when  the  blood  is  drawn. 

Finally,  a  few  delicate  threads  of  fibrin  may  be  observed 
stretching  in  different  directions  across  the  field  of  the  micro- 
scope ;  to  see  these  distinctly  a  good  lens  is  needed. 

Blood  on  the  warm  stage. — In  order  properly  to  study 
the  vital  phenomena  which  are  displayed  by  the  white  blood- 
corpuscles  it  is  necessary,  in  the  case  of  man  and  warm-blooded 
animals,  to  maintain  the  drop  of  blood  under  observation  at 
or  near  the  temperature  of  the  body.  For  this  purpose  we 
employ  what  is  known  as  a  warm  stage,  of  which  there  are 
several   forms   in   use.     The  simplest  consists  merely  of   an 


WARM   STAGE  53 

oblong  copper  platen  (fig.  3;")),  two  inches  l)y  one  inch,  from  one 
side  of  which  a  rod  of  the  same  metal,  four  or  five  inches  long, 
projects.  This  plate  has  a  round  aperture  in  the  middle,  half  an 
inch  in  diameter,  and  is  fastened  to  an  ordinary  slide  by  sealing- 
wax.  The  preparation  is  made  as  follows  : — Take  tirst  a  clean, 
long  piece  of  covering-glass  about  2  inches  by  1  inch,  which  in 
this  case  is  to  be  used  instead  of  a  slide,  and  on  it  make  a  pre- 
paration of  blood  oljtained  from  the  finger,  as  in  the  first 
preparation,  carefully  covering  with  an  ordinary  cover-glass. 
If  there  is  now  not  enough  blood  to  fill  the  space  between  the 
two  glasses,  add  a  little  salt  solution  at  one  edge  of  the  smaller 
cover-glass  ;  but  if,  on  the  other  hand,  there  is  too  much,  soak  up 

Fig.  35 


Simple  warm  !^tage,  with  preparation  upon  it  enclosed  between  two  cover- 
glasses 

the  excess  with  a  small  piece  of  blotting-paper.  A  very  small 
camel-hair  pencil  which  has  been  dipped  in  olive-oil  is  now  to 
be  drawn  gently  along  each  edge  of  the  smaller  glass  :  this 
will  prevent  evaporation  from  the  edges,  which  would  other- 
wise quickly  ensue  on  warming  the  preparation.  The 
glass  slide  which  bears  the  copper  plate  having  been  clamped 
on  to  the  microscope  stage,  with  pieces  of  cloth  underneath 
it  to  prevent  the  heat  from  being  conducted  away  by  the 
metal  stage,  the  preparation  thus  made  is  placed  upon  the 
plate,  and,  having  been  brought  in  focus,  one  or  more  white 
corpuscles  are  selected  for  observation — a  high  magnifying 
power  being  used.     The  rod  is  now  heated  near  its  end  by  a 


54 


PEACTICAL  HISTOLOGY 


small  spirit-lamp,  and  the  heat  is  conducted  by  the  rod  to  the 
copper  plate,  and  from  this  is  transmitted  to  the  preparation. 
A  fragment  of  some  fatty  substance  such  as  paraffin  or  a 
mixture  of  wax  and  cacao-butter,  melting  at  about  35°  C,  is 
placed  near  one  edge  of  the  preparation,  and  another  fragment, 
melting  at  about  40°  C,  near  the  opposite  edge  (fig.  36).     The 

Fig.  36 


Simple  warming  apparatus,  complete,  shown  in  operation 

lamp  is  now  gradually  approached  along  the  rod  until  it 
arrives  at  a  spot  the  heat  transmitted  from  which  is  just 
sufficient  to  melt  the  one  fragment  but  not  the  other,  and 
it  is  then  left  burning  at  that  spot  ;  the  preparation  will 
then  be  maintained  at  a  temperature  approaching  that  of 
the  blood. 


WARM   STAGE  55 

Another  way  of  mounting  the  blood  is  in  the  form  of  a  tliin 
'hanging  drop'  on  tho  under  surface  of  the  cover-glass,  which  ip 
inverted  over  the  hole  in  the  copper  plate.  Before  putting  it  down, 
moisten  the  glass  slide  which  closes  the  hole  below  by  breathing 
on  to  it.  The  edges  of  tho  cover-glass  are  then  made  air-tight  by  a 
layer  of  oil  between  it  and  the  copper,  and  the  layer  of  blood, 
which  may,  if  desired,  have  been  diluted  with  salt  solution,  now 
liangs  within  a  shallow  moist  chamber.  The  advantage  of  adopting 
this  method  is  that  the  preparation  is  freely  exposed  to  the  air  of 
the  moist  chamber,  and  the  white  corpuscles  retain  their  activity 
for  a  longer  time  unimpaired. 

It  will  be  seen  that  as  the  preparation  begins  to  get  warm 
the  white  corpuscles,  which  were  perhaps  previously  rounded 
and  inert,  begin,  to  throw  out  processes  and  exhibit  amoeboid 
movements,  which  become  more  marked  as  the  temperature 
rises,  so  that  by  virtue  of  these  an  actual  change  of  place  from 
oiie  part  of  the  field  to  another  may  be  effected.  It  is  well  in 
making  this  observation  to  select  a  single  corpuscle  and  to 
sketch  its  outline  and  that  of  its  more  immediate  surroundings 
at  intervals  of  half  a  minute.  As  the  corpuscles  become 
spread  out  in  creeping  along  the  glass  one  or  more  nuclei  may 
sometimes  be  seen  indistinctly  within  them.  Clear  spaces  or 
vacuoles  are  also  to  be  seen  in  the  protoplasm  of  some,  whilst 
others  are  filled  with  granules,  which  stream  with  the  move- 
ments of  the  corpuscles. 

The  red  corpuscles  in  this  preparation  may  be  disregarded, 
for  they  show  no  trace  of  amceboid  movement.  The  slight 
shaking  movement  which  many  of  them  exhibit  is  the  molecular 
or  Brownian  movement  common  to  all  minute  solid  particles 
floating  in  a  liquid. 

After  the  observations  recorded  in  the  preceding  paragraph 
are  completed  the  action  of  an  excess  of  heat  may  be  observed  ; 
but  it  is  better  to  use  for  this  purpose  a  larger  apparatus,  in 
which  the  degree  of  heat  can  be  measured  by  a  thermometer. 
Such  a  one  is  shown  in  fig.  37.  In  this  the  preparation  is 
placed  upon  the  brass  box  a,  which  rests  on  the  stage  of  the 
microscope,  and  is  pierced  in  the  centre  by  a  tubular  aperture 


56 


PEACTIGAL  HISTOLOGY 


to  admit  light  to  the  object.  The  box  is  connected  by  india- 
rubber  tubes  with  a  hollow  metal  jacket,  f,  and  the  whole 
system  thus  constituted  is  completely  filled  to  the  exclusion  of 
air  with  water  previously  boiled  and  cooled.  The  water  is 
warmed  at  p'  by  a  small  gas-flame,  and  rising  through  the  tube 
c  communicates  its  heat  to  the  box  a,  the  temperature  of  which 
is  measured  by  a  small  thermometer,  &,  inserted  through  an 
obliquely  placed  tube  quite  into  the  central  hole  and  immer 
diately  under   the  preparation.     The  cooled  water  from  the 

Fig.  37 


Apparatus  for  maintaining  a  constant  temperature  under  the  microscope  ^ 

stage  descends  down  the  tube  c',  to  pass  again  round  to  the 
flame,  and  in  this  way  the  water  constantly  circulates.  The 
bulbed  tube  d,  filled  with  mercury,  serves  to  regulate  the  flow 
of  gas,  so  as  to  keep  the  temperature  constant  at  any  desired 
point.  This  is  effected  by  turning  the  steel  screw  e,  when  this 
point,  whatever  it  may  be,  is  reached,  so  as  to  raise  the 
mercury  in  the  glass  tube,  and  thus  almost  block  up  the  lower 
end  of  a  small  steel  or  glass  tube  which  is  fixed  into  the  upper 
1  See  Quarterly  Journal  of  Microscopic  Science,  1875,  vol.  xiv. 


WARM  STAGE  57 

end  of  the  tube  d.  The  gas  passes  through  the  small  tulx; 
and  then  above  the  mercury  and  between  the  two  tubes,  to  Ije 
conducted  by  the  side  piece  A  to  the  burner  below  ;  and  it  will 
be  understood  that  if  the  temperature  now  rises  higher  in  the 
reservoir  /i  which  surrounds  the  mercury,  this  on  being  warmed 
will  expand  and  tend  to  cut  t)flF  more  of  the  gas,  and  thus 
reduce  the  flame,  on  which  the  mercury  will  again  contract, 
and  the  flame  will  rise  in  consequence,  and  so  on.  It  is  found 
that  an  equilibrium  soon  becomes  established,  and  the  tem- 
perature of  the  water  and  stage  remains  almost  absolutely 
constant.  To  raise  or  lower  the  temperature  all  that  is 
required  is  to  screw  out  or  in  the  screw  e.  The  small  included 
tube  is  pierced  with  a  minute  aperture,  to  allow  a  constant 
passage  of  gas,  so  as  to  prevent  the  flame  from  being 
extinguished  in  the  event  of  the  complete  occlusion  by  the 
mercury  of  the  lowei*  end  of  the  tube  in  question. 

It  will  be  found  that  up  to  and  a  little  beyond  the  normal 
temperature  of  the  blood  the  white  corpuscles  become  moro 
active  in  their  movements,  but  on  gradually  warming  the  pre- 
paration still  more,  a  point  (50°  C.)  is  reached  at  -svhich  they 
draw  in  their  processes,  become  spherical,  and  show  no  longer 
any  signs  of  vitality,  the  temperature  being  now  sufficient  to 
kill  the  corpuscles.  The  red  corpuscles  remain  unaltered  until 
a  temperature  of  about  55°  C.  is  reached,  when  they  become 
altered  in  shape,  and  globular  ;  soon  they  also  begin  to  show 
alterations  of  outline  and  even  to  throw  out  beaded  processes, 
but  these  changes  are  not  due  to  any  vital  amoeboid  movements 
but  to  the  action  of  heat  in  softening  the  envelope  of  the 
corpuscles.  Eventually  this  envelope  gives  way  altogether, 
and  their  colouring  matter  is  then  discharged  and  becomes 
dissolved  out  in  the  surrounding  serum. 

Another  and  a  still  more  accmrate  method  of  observing  the 
efiect  of  varying  degrees  of  heat  upon  a  preparation  is  to  enclose 
tlio  whole  microscope,  except  the  upper  end  of  the  tube  and  the 
fine  adjustment,  in  a  metal  box,  which  has  a  window  in  front  for 
admission  of  light  to  the  mirror,  and  a  gas  burner  and  regulator 
below  for  raising  the  temperature  of  the  air  within  the  box  (tig.  38). 


58 


PRACTICAL  HISTOLOGY 


Action  of  Reagents  upon  the  Blood. — The  red  cor- 
puscles in  the  two  preceding  preparations  appeared,  even 
under  the  highest  power,  perfectly  homogeneous  and  structure- 

FiG.  38 


Method  of  enclosing  a  microscope  within  a  warm  box 

less  ;  but  it  can  be  shown,  by  the  application  of  reagents  to  the 
blood  under  the  microscope,  that  they  in  reality  consist  of  two 
separable  parts — the  envelope  (formed  of  various  chemical  com- 
pounds, such  as  lecithin,  cholesterin,  nucleo-proteid),  which 


THE   BLOOD  59 

is  colourless  iind  gives  the  shape  to  the  whole  corpuscle,  and 
the  coloured  contents,  which  consist  chiefly  or  wholly  of  a 
solution  of  a  red  crystallisable  substance,  haemoglobin.  The 
mode  of  application  of  reagents  is  as  follows  : — A  drop  of  blood 
is  got  ready  as  in  the  first  preparation,  and  whilst  under  obser- 
vation a  small  drop  of  the  reagent  (which  should  as  a  rule  be 
freshly  prepared)  is  allowed  to  come  into  contact  with  the  edge 
of  the  cover-glass.  Some  of  the  fluid  flows  under  this  and 
mixes  with  the  drop  of  blood  ;  the  current  produced  by  it  at 
first  drives  the  corpuscles  before  it,  but  they  soon  become 
stationary,  and  then  the  part  of  the  preparation  should  be 
selected  for  oliservation  where  the  reagent  is  gradually  ditifusing 
itself  amongst  the  corpuscles.  In  this  way  every  stage  in  its 
action  may  readily  be  studied. 

Action  of  water. — When  a  drop  of  distilled  water  is  applied 
in  the  manner  above  described  the  first  effect  is  seen  to  be  that 
the  red  corpuscles  begin  to  lose  their  discoid  form,  first  one  of 
their  sides  becoming  bulged  out  so  that  they  ai'e  cup-shaped, 
and  then  the  other  side,  so  that  they  are  now  completely 
globular,  as  may  be  seen  when  they  roll  over.  Meanwhile  the 
haBmoglobin  is  being  dissolved  out  of  the  corpuscles  by  the 
Avater,  so  that  they  are  soon  quite  colourless  and  hardly  to  be 
detected  in  the  now  reddish  fluid.  Some  seem  to  offer  greater 
resistance  to  the  action  of  the  water  (and  indeed  of  most  re- 
agents) and  to  retain  their  colouring  matter  longer  than 
others. 

The  white  corpuscles  are  also  soon  affected.  They  cease 
their  ama^boid  movements  and  begin  to  swell  up  by  imbibition 
of  fluid,  whilst  at  the  same  time  with  a  high  power  the 
granules  in  their  interior  may  be  seen  to  exhibit  the  dancing 
movement  which  is  characteristic  of  minute  particles  floating 
in  liquid.  Often  the  corpuscles  present  clear  bulgings  at  their 
circumference,  or  their  substance  may  appear  to  burst  at  one 
point  and  become  diflused  in  the  water.  As  they  swell  and 
become  clearer,  the  nuclei  generally  come  into  view,  and 
soon   these  also  become  swollen,   and   with   the  rest  of   the 


60  PKACTICAL  HISTOLOGY 

corpuscles  eventually  disintegrate,  nothing  being  left  but  a 
few  granules. 

Water  is  thus  proved  to  have  a  characteristic  action  upon 
the  protoplasmic  white  corpuscles  as  well  as  upon  the  very 
easily  alterable  red  discs,  and  this  fact  must  be  borne  in  mind 
in  investigating  the  action  of  reagents  or  poisonous  substances, 
both  upon  the  blood-corpuscles  and  upon  the  tissues  generally. 
If  a  reagent  is  to  be  employed  in  weak  solution,  therefore,  it 
is  w^ell  to  dissolve  it  either  in  salt  solution  or  in  fresh  serum  ^ 
instead  of  water.  Washing  with  water  tissues  which  are 
subsequently  to  be  submitted  to  microscopical  examination 
is  for  a  similar  reason  to  be  deprecated  ;  but  if  a  trace  of 
bichromate  of  potash  or  of  chromic  acid,  or  a  little  common 
salt,  be  previously  added  to  the  water,  its  deleterious  effect 
is  in  great  measure  obviated. 

Action  of  acids. — To  investigate  the  action  of  dilute 
acids  it  is  best,  as  just  explained,  to  mix  the  acid  with  salt 
solution  instead  of  water  :  1  part  of  glacial  acetic  acid  to  200 
of  salt  solution  is  an  appropriate  strength  for  the  blood.  The 
preparation  is  made  in  the  usual  way,  and  the  drop  allowed  to 
run  in  at  the  edge  of  the  cover-glass.  The  action  of  the  weak 
acid  upon  the  red  corpuscles  is  seen  to  be  quite  like  that  of 
water  :  they  are  first  rendered  globular  and  then  decolourised. 
Upon  the  white  corpuscles  it  has  a  somewhat  different  action, 
for  although  the  protoplasm  of  the  corpuscle  becomes  partly 
swollen  out  into  a  clear  spheroid,  the  nuclei  are  not  swollen 
by  the  reagent,  but  are  brought  very  distinctly  into  view, 
and  remain  usually  at  one  side  of  the  corpuscle,  with  a  little 
granular  matter  pi-ecipitated  around  them. 

Action  of  tannic  acid, — The  action  of  tannic  acid  upon  the 
red  corpuscles  is  peculiar  and  interesting.  Like  other  acids  it 
tends  to  cause  the  coloured  part  of  the  red  corpuscle  to  pass 
out  from  the  envelope  ;  but  as  the  coloured  material  is  exuding 
it  becomes  coagulated  by  the  astringent  reagent,  and  in  place 

^  The  serum  employed  should  be  from  the  blood  either  of  the  same  animal 
or  of  one  belonging  to  the  same  species. 


MOIST  CHAMBER  01 

of  being  dissolved  in  the  surrounding  ]i(iuid,  as  after  the  action 
of  acetic  acid,  it  remains  attached  to  the  envelope  as  a  small, 
bright,  reddish  projection. 

In  the  first  part  of  the  reaction — viz.  the  rendering  the  cor- 
puscles globular — tannic  acid  acts  similarly  to  other  weak  acids. 

The  most  convenient  strength  of  solution  to  use  is  1  per 
cent.  ;  it  should  be  freshly  prepared.  It  is  well,  moreover, 
first  to  mix  the  drop  of  blood  upon  which  it  is  desired  to  test 
the  action  of  the  reagent  with  an  equal  amount  of  salt  solu- 
tion ;  otherwise  the  tannic  acid  produces  such  a  dense  pre- 
cipitate with  the  albumin  of  the  serum  that  the  view  of  the 
corpuscles  is  greatly  obscured,  and  indeed  the  reagent  with 
difficulty  reaches  thorn. 

Action  of  alkalies. — A  mixture  of  1  part  of  caustic 
potash  to  500  of  salt  solution  may  be  used,  a  drop  being  added 
to  the  preparation  in  the  usual  way  (p.  59).  The  reaction 
takes  place  very  suddenly  ;  the  corpuscles,  both  white  and  red, 
swell  up  as  soon  as  the  reagent  reaches  them,  appear  to  burst, 
and  then  entirely  disappear.  The  white  are  affected  by  a 
weaker  solution  than  the  red.  This  is  apparent  from  the  fact 
that,  as  the  liquid  slowly  diffuses  and  mixes  with  the  blood, 
the  white  may  be  seen  to  become  destroyed  in  parts  where  the 
red  are  still  unaffected. 

Chloroform, — To  observe  the  action  of  chloroform  vapour 
upon  blood  a  moist  chamber  is  used.  This  is  an  apparatus  for 
keeping  a  tissue  or  fluid  under  examination  in  its  naturally 
moist  condition,  whilst  at  the  same  time  allowing  its  surface 
to  be  exposed  to  air  or  to  any  desired  gas  or  vapour.  The 
simplest  form  of  moist  chamber  is  made  of  a  small  piece 
of  soft  putty  or  modelling-wax,  which  has  been  rolled  out 
between  the  fingers  into  a  round  cord  about  2  inches  long  and 
^  inch  thick  ;  the  ends  of  the  cord  are  united  so  as  to  form  a 
ring,  and  this  is  placed  on  the  middle  of  a  clean  glass  slide.  A 
drop  of  water  is  put  at  the  bottom  of  the  chamber  ;  this  is  for 
the  sake  of  keeping  the  atmosphere  of  the  chamber  moist ;  but 
the  object  may  be  equally  well  effected  by  breathing  into  the 


62 


PEACTICAL  HISTOLOGY 


space  as  it  is  being  covered  over.  The  object  is  prepared  on 
the  centre  of  a  clean  cover-glass,  which  is  then  inverted  over 
the  ring,  so  that  the  preparation  is  dependent  into  the  chamber, 
and,  whilst  freely  exposed  to  the  air  in  this,  is  entirely  pro- 
tected from  evaporation  and  may  be  readily  examined  through 
the  cover-glass,  to  the  under  surface  of  which  it  remains 
adhei-ent  (fig.  39). 

To  investigate  the  action  of  chloroform  vapour  it  is  neces- 
sary to  have  some  means  of  passing  this  into  the  moist  cham- 
ber whilst  the  drop  of  blood  is  under  observation.     For  this 

Fig.  39 


purpose  a  slide  is  employed  (fig.  40),  to  which  a  piece  of 
small  glass  tubing  has  previously  been  fixed  by  means  of 
sealing-wax.  This  is  done  by  heating  the  slide,  dropping  the 
sealing-wax  upon  it  while  hot,  then  heating  the  glass  tube  and 
laying  it  in  the  sealing-wax  upon  the  slide.  The  ring  of  putty 
is  so  placed  as  to  include  the  end  of  the  tube,  a  small  hole 
being  made  in  the  ring  to  afford  an  exit  for  the  current  of  air 
containing  the  vapour.  The  slide  is  then  clamped  on  to  the 
microscope  stage  (as  in  fig.  46,  p.  76),  and  the  glass  tube  is  con- 
nected by  indiarubber  tubing  to  a  bottle  containing  a  few 
drops  of  chloroform  and  furnished  mth  a  second  tube,  through 
which  air  can  be  blown. 


ENUMERATION   OF   ULOOD-CORPUSCLES 


63 


Before  the  cover-glass  is  superposed  the  blood  should  tirst 
have  been  spread  out  upon  it  into  a  thin  layer,  so  that  the 
chloroform  vapour  may  readily  act  upon  all  parts. 

Everything  being  thus  prepared,  and  some  of  the  blood- 
corpuscles  having  been  brought  clearly  under  observation, 
air  is  blown  gently  into  the  bottle,  and  passing  through  it 
becomes  charged  with  the  vapour  of  chloroform,  which  is 
conveyed  by  the  tube  into  the  moist  chamber,  where  it  acts 
upon  tlie  layer  of  blood  which  is  on  the  under  surface  of  the 
cover-glass.     After   a   short   time   it   will   be   seen  that  the 

Fig.  40 


Chamber  for  passing  a  gas  or  vapour  over  a  preparation  under  the  microscope 

amoeboid  movements  of  the  pale  corpuscles  are  arrested, 
and  that  the  red  corpuscles,  as  under  the  action  of  water 
and  dilute  acids,  become  globular  ;  subsequently  their 
haemoglobin  becomes  dissolved  and  discharged  out  in  the 
serum.  It  will  further  be  observed,  both  in  this  and  in  the 
other  preparations  in  Avhich  this  change  has  taken  place  in 
the  red  corpuscles,  that  to  the  naked  eye  the  blood  has  changed 
from  scarlet  to  lake,  and  that  whereas  when  the  corpuscles 
were  intact  even  a  thin  layer  of  blood  presented  a  somewhat 
opaque  appearance,  it  is  now  completely  transparent.  Hence 
we  may  infer  that  the  opacity  of  the  unaltered  blood  is  due  to 
the  presence  of  the  red  particles. 

Enumeration    of    blood-corpuscles. — The    following    are 
necessary  for  counting  the  blood-corpuscles,     (a.)  A  diluting 


64  PKACTICAL  HISTOLOGY 

solution.     Either  that  recommended  by  Hayem  may  be  used, 
viz.  : — 

Distilled  water  .         .  200  cc.  Common  salt     .         .       1  grm. 

Sulphate  of  soda         .       5  grm.       Corrosive  sublimate  .    0*5  grm. 

or  Sherrington's  fluid,  viz.  : 

Distilled  water 300  cc. 

Coramon  salt         .         .         .         .         .     1'2  grm. 
Neutral  pot.  oxalate       ....     1"2  grm. 

to  which  eosin  may  be  added  if  desired. 

In  the  latter  fluid,  if  means  be  taken  to  prevent  evaporation, 
the  white  corpuscles  remain  living  and  amoeboid  for  days,  and 
the  granules  become  stained  in  vivo. 

(b.)  A  measuring  pipette  holding  10  cubic  millimetres, 
and  about  1  centimetre  long.  Thia  is  made  out  of  a  piece  of 
thick-walled  capillary  tube,  and  is  ground  and  polished  to  a 
blunt  point  at  one  end.  It  has  a  flat  narrow  band  of  German 
silver  attached  to  its  middle,  to  serve  as  a  handle  (fig.  42,  a). 

(c.)  A  cylindrical  mixing  vessel  marked  to  be  filled  up  to 
either  50  or  100  times  the  capacity  of  the  pipette. 

Fig.  41 


Slide  ruled  in  squares  of  "1  mm.  each,  for  enumerating  blood  corpuscles 
under  the  microscope 

(d.)  A  counting  slide,  marked  at  its  centre  by  a  diamond 
with  lines  forming  squares  of  O'l  millimetre,  and  having  a 
ground-glass  ring  cemented  to  it  which  is  exactly  either  0-1  or  0-2 


ENUMEJIATION   OF    IJLOOD-COIIPUSCLES  65 

millimetre  thick  (Gowers).  The  slide  may  be  fitted  with 
spiings  to  press  the  cover-glass  down  firmly  on  the  ring. 

(e.)  A  small  glass  stirrer,  needles,  worsted,  cover- glasses, 
and  a  dropping  tube  (fig.  42,  b). 

Proceed  as  follows  : — See  that  all  the  apparatus  is  clean 
and  dry.  (The  capillary  pipettes  can  be  readily  dried  by  pass- 
ing worsted  through  them  with  a  needle.)  Prick  the  finger 
squeezing  it  slightly  to  force  the  blood  out  more  quickly,  and 
when  a  large  enough  drop  has  exuded,  place  the  point  of  a 
capillary  pipette  in  it,  and  allow  the  pipette  to  fill  completely 
with  blood,  which  it  will  easily  do  by  virtue  of  its  capillarity. 
Wipe  ofi'  with  the  finger  any  blood  which  may  have  got  on  the 
outside  of  the  capillary.  Hold  the  capillary  ovei-  the  mixing 
vessel,  and  from  the  dropping  tube  let  some  of  the  diluting 
solution  fiow  thi'ough  the  capillary,  thus  completely  washing 
out  the  blood,  until  the  mixer  is  filled  up  to  the  100  mark. 
The  blood  is  thereby  diluted  to  form  1  per  cent,  of  the 
mixture.  If  it  be  desired  to  form  a  2  per  cent,  mixture,  the 
vessel  must  only  be  filled  to  the  50  mark.  This  is  a  better 
degree  of  dilution  when  the  white  corpuscles  are  to  be  enume- 
rated as  well  as  the  red.  Now  stir  the  mixture  and  place  a 
drop  of  it  upon  the  counting  slide,  so  that  on  placing  the 
cover-glass  upon  the  ring  it  (the  cover-glass)  comes  in  con- 
tact with  the  top  of  the  drop.  Assuming  the  ground-glass  ring 
to  be  0-1  millimetre  thick,  the  mixture  will  then  form  a  layer 
0*1  millimetre  thick  between  the  cover-glass  and  slide,  and  the 
liquid  above  each  square  will  be  0*1  millimetre  cube.  In  a 
few  minutes  the  blood-corpuscles  will  have  sunk  to  the  bottom, 
and  will  rest  on  these  squares,  where  they  may  be  counted. 
The  number  on  each  square  represents  the  number  in  O'OOl 
(7-„\,yth)  cubic  millimetre  of  the  mixture.  To  arrive  at  the 
number  in  1  cubic  millimetre  it  is  best  to  count  the  number  on 
ten  squares  and  multiply  by  100.  The  product  multiplied  by 
100  or  by  50  (according  as  a  1  per  cent,  or  2  per  cent,  mixture 
was  used)  gives  the  number  in  a  cubic  millimetre  of  undiluted 
hlood.     Of  course  the  number  on  ten  squares  may  be  at  once 

p 


66  PEACTICAL  HISTOLOGY 

multiplied  by  10,000  or  by  5,000,  as  the  case  maybe.  It  will  be 
found  that  in  normal  human  blood  (male)  there  are  on  an  average 
5,000,000  red  corpuscles  to  the  cubic  millimetre.  For  the  white 
corpuscles  it  is  desirable  to  count  the  number  on  a  much 
larger  number  of  squares. 

Oliver's  method. — A  much  more  ready  and  at  least  equally 
accurate  method  of  estimating  the  number  of  coloured  cor- 
puscles in  blood  is  that  devised  by  Dr.  G.  Oliver.  A  small 
quantity  of  blood  is  taken  up  as  before  into  a  short  pipette 
(fig.  42,  a),  and  at  once  washed  out  of  this  by  the  dropping 
tube,  6,  into  a  graduated  flattened  test  tube  (c),  with  Hayem's 
diluting  mixture  (which  must  not  contain  colouring  matter). 
The  graduations  of  the  tube  are  so  adjusted  to  the  capacity 
of  the  pipette  that  with  normal  blood  (assumed  to  contain 
5,000,000  red  corpuscles  to  the  cubic  millimetre)  the  light  of  a 
small  wax  candle  placed  at  a  distance  of  three  yards  from  the 
eye  in  a  dark  room  is  just  transmitted  as  a  fine  bright  line 
when  looked  at  through  the  tube  held  edgeways  between  the 
fingers  (d)  and  filled  up  to  the  100  mark  of  the  graduation- 
If  there  are  fewer  corpuscles  than  the  normal,  less  of  the 
diluting  solution  is  required  for  the  light  to  be  transmitted ;  if 
more  than  normal,  more  of  the  solution  must  be  added.  The 
tube  is  graduated  above  and  below  the  100  mark  so  as  to  indi- 
cate in  percentages  every  decrease  or  increase  of  corpuscles  per 
cubic  millimetre,  as  compared  with  the  normal  standard  of  100 
per  cent.  By  this  means  an  accurate  result  can  be  obtained  in 
two  or  three  minutes,  whereas  by  the  most  expeditious  observer 
an  actual  enumeration  will  take  from  10  to  15  minutes.^ 

Examination  of  frog's  or  newt's  blood. — The  simplest  way 
to  obtain  frog's  blood  for  examination  is  to  cut  ofl'  the  tip  of 
one  of  the  digits,  having  previously  wiped  it  dry  with  a  cloth, 
and  to  collect  upon  a  clean  cover-glass  the  small  drop  of 
reddish  fluid  which  exudes.  The  glass  is  then  inverted  upon 
a  slide  and  the  drop  is  examined.  The  blood  so  obtained  is 
mixed  with  lymph,  and  the  corpuscles  are  consequently  less 

1  Oliver's  ai3j)ai'atus  is  supplied  by  the  Tintometer  Company,  6  Farringdon 
Avenue,  E.G. 


ENUMERATION    OF   BLOOD-COKI'USCLE.S  67 


B^io.  42 


Oliver's  apparatus  for  rapidlj-  estimating  the  number  of  corpuscles  in  a  sample 
of  blood  by  means  of  the  opacity  method 


f2 


68  PKACTICAL   HISTOLOay 

crowded  and  better  adapted  for  observation  than  when  the 
blood  is  undiluted.  For  newt's  blood  the  end  of  the  tail  is 
cut  off,  and  a  drop  of  blood  similarly  obtained.  To  procure 
blood  unmixed  with  lymph  the  animal  should  be  pithed,  and 
laid  upon  its  back.  The  heart  is  then  exposed  and  snipped 
with  scissors,  and  a  small  drop  of  the  blood  which  exudes  is 
taken  up  with  a  glass  rod,  transferred  to  a  slide  and  covered. 
But  before  placing  the  cover-glass  down  in  the  usual  way  a 
small  length  (^  inch)  of  a  delicate  hair  should  be  placed  in  the 
drop,  so  that  when  the  cover-glass  settles  down,  the  corpuscles, 
here  comparatively  large,  may  not  be  crushed  by  its  weight. 

When  the  preparation  obtained  from  either  of  these  sources 
is  examined  it  will  at  once  be  seen  that  the  coloured  cor- 
puscles are  larger  and  fewer  in  number  than  in  human  blood, 
and  do  not  tend  to  form  rouleaux,  although  they  adhere  to- 
gether in  a  less  regular  fashion  ;  that  they  have  an  elliptical 
outline  when  lying  flat,  but  when  seen  edgeways  look  quite 
narrow  and  pointed  at  the  ends,  with  a  slight  and  gradual 
bulging  at  either  side  ;  so  that,  although  disc-shaped,  like  the 
mammalian  blood-corpuscle,  so  far  from  being  biconcave, 
they  are  biconvex.  The  bulging  is  due  to  the  pi'esence  in 
the  middle  of  the  corpuscle  of  another  part  besides  the 
envelope  and  the  coloured  contents  found  in  the  mammalian 
disc.  This,  the  mtcleits,  can  readily  be  made  out  in  most  of 
the  corpuscles  as  a  somewhat  elongated,  colourless,  and  slightly 
granular  elliptical  body,  about  a  third  the  length  of  the  cor- 
puscle, and  often  lying  not  quite  in  the  middle  but  somewhat 
excentrically.  Occasionally  the  nucleus  is  seen  to  be  round  ; 
but  this  is  an  accidental  change,  and  may  be  brought  about 
by  mechanical  injury.  Indeed,  if  the  precautions  above  re- 
commended for  avoiding  pressure  are  not  taken,  a  large 
number  of  the  corpuscles  become  injured,  so  much  so  as  even 
to  be  ruptured  and  destroyed  altogether,  in  which  case  the 
rounded  nuclei  are  liberated,  and  may  be  mistaken  by  the 
beginner  for  pale  blood-corpuscles. 

With  regard  to  the  white  corpuscles,  it  will  be  observed 


FEEDING    OV   AVIIITK   CORPUSCLES  69 

that  tliey  are  more  numerous  and  larger  than  in  human 
blood.  Moreover,  they  soon  begin,  even  in  the  cold,  to  exhibit 
\'ery  distinct  amaboid  movements  ;  and,  on  account  of  the 
ifi-eater  size  of  tlie  corpuscles,  both  these  and  the  other 
phenomena  exhibited  by  them  are  much  more  striking,  and 
these  corpuscles  are  therefore  much  better  suited  for  observa- 
tion than  those  of  mammals,  which  in  other  respects  they 
closely  resemble.  The  distinction  between  the  tincly-  and  the 
coarsely -granular  corpuscle  is  met  with  again  here  ;  but  there 
is  also  sometimes  seen  a  third  kind  of  corpuscle,  fusiform  or 
oat -shaped  and  devoid  of  ania'l)oid  properties. 

Feeding  of  the  white  corpuscles. — The  white  corpuscles 
of  the  blood  exhibit  a  strong  tendency  to  take  into  their  in- 
terior any  small  particles  of  solid  material  which  may  happen 
til  be  in  their  neighbourhood.  This  tendency  they  ha\-e  in  com- 
mon with  all  amoeboid  organisms,  and  it  is  nowhere  better 
seen  than  in  the  case  of  the  amoeba  itself.  Because  of  their 
greater  size,  number,  and  activity,  it  is  better  to  take  the  newt's 
corpuscles  for  this  experiment  than  those  of  the  mammal. 

Any  substance  consisting  of  fine  insoluble  particles,  such  as 
\  ermilion  or  Chinese  ink,  may  be  rubbed  up  with  salt  solution 
and  used  for  this  experiment,  but  the  most  convenient  material 
is  yeast.  A  very  small  fragment  of  German  yeast  is  rubbed  up 
with  a  little  salt  solution  so  that  the  solution  is  faintly  milky. 
A  small  drop  of  this  mixture  is  then  placed  on  a  slide,  and  a 
drop  of  blood  is  added  to  and  well  mixed  up  with  it.  The 
preparation  is  then  covered,  and  the  edges  of  the  cover-glass 
are  painted  with  a  film  of  oil  to  preclude  evaporation.  If  the 
white  corpuscles  are  now  observed,  it  will  be  seen  that  certain 
of  them  gradually  take  into  their  substance  the  yeast  particles 
with  which  they  happen  to  come  in  contact  in  their  amoeboid 
movements.  On  careful  watching  it  may  be  made  out  that 
the  process  of  inception  commences  by  the  throwing  out  of 
processes  which  surround  the  particle  to  be  taken  in,  and 
meet  and  coalesce  beyond  it  ;  once  included  in  this  manner, 
the  granule  afterwards  becomes  gradually  carried,  presumably 


70  PEACTICAL   HISTOLOGY 

by  the  movements  of  the  protoplasm,  more  towards  the  centre. 
If,  after  observing  the  preparation  in  this  way  for  half  an 
hour,  it  be  laid  aside  for  two  or  three  hours,  it  will  be  found 
at  the  expiration  of  that  time  that  many  of  the  white  cor- 
puscles have  taken  in  a  large  number  of  the  yeast  particles, 
for  they  do  not  discharge  their  cargo,  but  carry  it  about  in 
their  movements  from  place  to  place. 

It  will  be  found  that  it  is  the  large  corpuscles  of  the  finely 
granuled  kind  which  exhibit  this  tendency  to  the  most  marked 
extent,  the  corpuscles  with  coarse  granules,  although  amoeboid, 
are  not  phagocytes. 

Migration  of  white  corpuscles. — The  process  of  migration 
can  be  quite  easily  seen  in  progress  in  transparent  parts  of 
animals,  and  will  be  afterwards  studied  when  the  methods  of 
observing  the  circulation  of  the  blood  are  described.  But  for 
exhibiting  the  active  migration  of  the  white  blood-corpuscles 
nothing  is  more  striking  than  the  examination  of  a  capillary 
glass  tube  in  which  frog's  blood  has  been  collected  and  has 
coagulated.  A  high  power  is  to  be  employed,  and  the  capillary 
tube  must  therefore  be  very  fine  ;  and  in  order  that  the  wall  of 
the  tube  should  be  as  thin  as  possible,  it  must  be  drawn  out  from 
a  piece  of  large  and  thin  tubing.  The  capillary  tube  is  filled  with 
frog's  or  newt's  blood,  except  near  the  ends  ;  these  are  then 
sealed  by  holding  them  successively  in  the  flame  for  a  second  ; 
the  tube  is  then  placed  in  a  drop  of  cedar  oil  or  thick 
Canada  balsam  on  a  slide,  covered  with  a  thin  glass,  and  at 
once  examined.  The  object  of  the  essential  oil  is  to  correct 
in  some  measure  by  its  high  refracting  power  the  effect  upon 
the  light  of  the  cylindrical  glass  tube.  After  a  few  minutes 
the  clot  is  seen  to  be  getting  smaller,  and  a  layer  of  clear 
serum  collects  between  it  and  the  glass  ;  the  quantity  of  this 
gradually  increases,  and  soon  portions  of  white  corpuscles 
begin  to  project  from  the  surface  of  the  clot.  These  protrude 
more  and  more,  and  others  make  their  appearance,  and  all 
begin  to  throw  out  numerous  amceboid  processes,  which  are 
actively    advanced    and    reti-acted.      By   aid   of    these   the 


ACTION   OF  WARMTH  AND  ELECTRIC  SHOCKS        71 

corpuscles   gradually  emerge   from    the   shi-inking   clot,    and 
eventually  become  free  in  the  surrounding  serum  (fig.  43). 

Influence  of  warmth  on  white  corpuscles  of  Amphibia. — 
The  action  of  gentle  warmth  in  accelerating  the  movements  of 
the  pale  corpuscles  of  the  frog  or  newt  may  be  investigated 
with  the  same  apparatus  as  was  used  for  the  observation  of 
mammalian  blood  at  the  temperature  of  the  body.    But  it  will 

Fig.  43 


White  corpuscles  of  frog's  blood  migTuting  from  clot.    HigUy  magnified.    The 
clot  has  shrunk  considerably  from  the  sides  of  the  capillary  tube 


be  found  that  if  the  temperature  be  allowed  to  rise  so  high  as 
38°  C.  the  movements  of  the  corpuscles  of  these  cold-blooded 
animals  will  soon  permanently  cease,  the  corpuscles  being 
killed  (heat-rigor).  So  that  unless  this  result  is  desired,  a 
paraffin  of  lower  melting-point  must  be  employed  to  indicate 
the  temperature  limit  which  is  not  to  be  exceeded. 

Action  of  electric  shocks. — For  this  ;i  slide  must  be  spe- 
cially prepared  by  cementing  to  its  upper  surface  with  shellac 


72 


PKAGTICAL  HISTOLOGY 


varnish  two  slips  of  gold-leaf  or  tinfoil,  with  pointed  ends 
which  almost  meet  in  the  middle  of  the  slide  (fig.  44,  a).  A 
drop  of  blood  is  put  here,  the  cover-glass  is  placed  over  it,  and 
the  portion  of  blood  which  lies  between  the  points  is  brought 
under  observation.     Or  a  moist  chamber  may  be  employed, 


Glass  slide,  witli  two  strips  of  tin- foil,  one  of  which  passes  round  to  the  under 
surface,  where  it  rests  on  the  brass  stage  of  the  microscope ;  the  other  strip 
is  isolated  from  the  stage,  and  may  be  connected  to  the  outer  coating  of  a 
Leyden  jar,  the  charge  of  which  is  made  to  pass  between  the  points  by 
connecting  the  knob  of  the  jar  with  the  brass-work  of  the  microscope. 
Opposite  a,  a  small  piece  of  the  foil  is  fixed  to  the  under  surface  of  the 
slide,  so  that  this  end  shall  be  level  with  the  other 


the  cover -glass  used  having  previously  had  two  strips  of  tin- 
foil cemented  to  it  (fig.  44,  b).  The  drop  of  blood  being  spread 
out  in  a  thin  layer  between  their  points,  is  quickly  inverted 
over  the  ring  of  putty  and  brought  under  observation.  The 
tinfoil  slips   are   kept   isolated  from   the   brasswork  of   the 


ACTION  OV  ELECTRIC  SHOCKS  ON  BLOOD     73 

microscope,  and  arc  so  arranged  that  the  charge  of  a  small 
Leyden  jar,  or  an  induced  current  of  electricity,  can  be  passed 
through  them  at  any  moment  (fig.  45).  One  or  more  ama^ljoid 
white  corpuscles  which  happen  to  be  in  the  path  which  the 
spark  must  take  in  tra\ersing  tlie  interval  between  the  points 
are  kept  in  view,  and  the  spark  is  then  allowed  to  pass.  The 
white   corpuscles  immediately  cease  moving,  withdraw  their 

Fifi.  45 


A  ppai-iitus  for  passing  electric  shocks  through  a  di'op  of  blood,  which  is  to  be 
examined  in  a  moist  chamber.  The  tin-foil  slips  are  cemented  near  their 
points  to  the  under  surface  of  the  cover-glass,  and  their  free  ends  are 
clamped  to  isolated  metal  supports,  connected  by  wires  to  an  induction-coil. 
Tlie  tin-foil  slips  are  isolated  from  the  brass  stage  of  the  microscope  by  the 
glass  slide  on  which  they  rest 


processes,  and  become  rounded  in  shape  ;  in  fact,  undergo 
general  contraction.  But  if  only  one  slight  shock  be  given 
they  soon  recover  and  resume  their  movements,  although  these 
are  often  somewhat  altered  in  character.  The  red  corpuscles 
are  but  slightly  if  at  all  aftected  ;  but  if  a  succession  of  shocks 
are  transmitted  from  an  electric  machine  or  an  induction-coil, 


74  PKACTICAL  HISTOLOGY 

electrolytic  action  is  set  up  in  the  fluid,  bubbles  of  gas  are 
developed,  the  effects  respectively  of  acids  and  alkalies  are  set 
up  in  the  neighbourhood  of  the  tinfoil  points,  and  the  red 
corpuscles  undergo  changes  brought  about  by  these. 

Presence  of  glycogen. — Many  of  the  white  corpuscles  con- 
tain a  certain  amount  of  glycogen,  either  in  distinct  granules 
or  in  a  more  diffused  form.  This  substance  becomes  stained 
of  a  reddish  mahogany  colour  by  solution  of  iodine,  and  may 
thus  be  readily  detected,  both  here  and  elsewhere.  The  solu- 
tion to  be  used  is  made  by  dissolving  1  gramme  of  iodine 
in  100  cc.  of  water  which  contains  2  grammes  of  iodide  of 
potassium  in  solution. 

The  preparation,  preferably  of  frog's  or  newt's  blood,  is 
made  in  the  usual  way,  and  the  iodine  solution  added  at  the 
side  of  the  cover-glass.  The  red  corpuscles  are  stained  of  an 
intense  yellow,  but  are  otherwise  little  altered,  except  that 
the  nucleus,  which  remains  unstained,  becomes  globular,  and 
bulges  out  at  either  surface  of  the  corpuscle.  The  white  cor- 
puscles are  instantly  arrested  in  their  movements  and  killed, 
preserving  exactly  the  form  which  they  exhibited  when 
reached  by  the  iodine  solution.  Being  of  less  specific  gravity 
than  the  latter,  they  tend  to  float  on  it  ;  and,  if  the  layer  of 
fluid  is  thick,  must  be  sought  by  focussing  upwards  in  the 
stratum  immediately  under  the  cover-glass.  The  main  sub- 
stance of  the  corpuscle  is  uniformly  stained  of  a  deep  yellow, 
but  many  contain  groups  of  more  darkly  stained  granules,  and 
from  others  are  seen  to  exude  after  a  time  pellucid  drops  of 
varying  size,  which  become  tinted  of  a  pale  port  wine  colour, 
and  no  doubt  contain  glycogen. 

Action  of  reagents — water,  acids,  &c. — upon  the  frog's 
blood-corpuscles. — The  action  of  reagents  upon  the  white 
blood-corpuscle  of  the  frog  is  exactly  the  same  as  upon  the 
mammalian  white  corpuscle  ;  upon  the  red  corpuscle  it  is  in 
the  main  similar,  but  in  some  cases  a  little  different,  the 
differences  partly  depending  upon  the  presence  of  the  nucleus. 
Thus  ivater  passing  through  the  envelope,  after  causing  the 


ACTION  OF  REAGENTS  ON  FROG'S  BLOOD  75 

corpuscle  to  swell  up  and  both  it  and  the  nucleus  to  become 
spheroidal,  extracts  the  coloured  contents  of  tlie  corpuscle, 
which  usually  resumes  its  oval  shape  after  the  haemoglobin  has 
escaped  ;  dilute  acetic  acid  brings  the  nucleus  strongly  into 
view,  and  decolourises  the  rest  of  the  corpuscle  :  tannic  acid 
causes  the  coloured  part  to  be  exuded  from  the  corpuscle,  to 
the  envelope  of  which  it  generally  remains  attached  as  an 
irregular  curdled  mass  (sometimes  the  coloured  part  is  pre- 
cipitated around  the  nucleus,  and  then  the  two  may  be  ejected 
from  the  stroma  together)  ;  chloroform  vapour  causes  the  red 
corpuscles  to  become  decolourised,  and  arrests  the  movements 
of  the  white  corpuscles  ;  but  these,  if  not  acted  on  for  too 
long  a  time  or  by  too  strong  a  mixture  of  chloroform  and  air, 
are  resumed  on  replacing  the  vapour  by  pure  air.  All  these 
reagents  are  applied  in  precisely  the  same  manner  as  with 
mammalian  blood,  to  the  description  of  which  the  reader  is 
referred. 

The  observations  may  be  made  with  as  great  or  with 
greater  advantage  upon  the  blood-coi'puscles  of  the  newt, 
which  are  larger  than  those  of  the  frog.  The  blood  should 
preferably  be  obtained  directly  from  the  heart,  and  not  by 
merely  snipping  a  piece  off  the  tail  and  collecting  the  drop 
which  exudes,  for  in  this  case  it  is  very  apt  to  become  mixed 
with  the  acrid  secretion  from  the  cutaneous  glands.  There 
are  in  addition  two  reactions  for  which  newt's  blood  is 
particularly  well  adapted,  viz.  the  actions  respectively  of 
boracic  acid  and  carbonic  acid  upon  the  red  corpuscles. 

Action  of  boracic  acid. — The  boi'acic  acid  is  used  in  solu- 
tion in  water  (2  per  cent.),  and  the  preparation  of  blood 
having  been  made  in  the  usual  way,  with  or  without  addition 
of  salt  solution,  a  drop  of  the  boracic  acid  solution  is  placed  at 
the  edge  of  the  cover-glass  and  allowed  slowly  to  mingle  with 
the  blood.  If  the  first  stages  of  the  reaction  are  fortunately 
observed,  it  will  be  seen  that  the  coloured  part  of  the  corpuscle 
is  becoming  collected  towards  the  centre  of  the  corpuscle  and 
accumulated  around  the  nucleus,  often  remaining,  however,  at 


76 


PEACTICAL  HISTOLOGY 


first  adherent  here  and.  there  to  the  circumference  of  the 
corpuscle,  and  shrinking  away  at  the  intermediate  points,  so 
as  to  present  somewhat  of  a  stellate  figure.'  Bu.t  soon  it  is 
entirely  withdrawn  and  collected  around  the  nucleus,  which 
has  become  rounded,  and  is  nearly  concealed  by  the  colouring- 
matter.  The  corpuscle,  now  decolourised,  has  also  in  many 
cases  become  circular,  and  the  coloured  nucleus  is  generally 
shifted  to  one  side,  and  eventually  altogether  extruded. 
Another  even  more  ready  method  of  obtaining  this  reaction  is 
to  drop  the  blood  directly  into  the  solution  of  boracic  acid. 

Fig.  46 


Apparatus  for  passing  carbonic  acid  gas  over  a  preparation  under  the 
microscope 

6,  bottle  containiug  marble  and  hydrochloric  acid  ;  h',  wash-bottle  ;  t,  india-rubber  tube 
conducting  the  gas  to  the  stage,  s 

Action  of  carbonic  acid. — To  investigate  the  action  of 
carbonic  acid  gas  the  blood  must  be  prepared  in  a  moist 
chamber,  like  that  used  for  chloroform  vapour  (fig.  40).  If 
the  preparation  is  very  quickly  made  the  nucleus  in  many  of 

'  Water  and  various  other  reagents  may,  in  the  first  instance,  have  a 
somewhat  similar  effect. 


CRYSTALS   OBTAINABLE   VllOy]    BLOOD  77 

the  red  corpuscles  cannot  at  lirst  be  distinguished,  for  in  the 
entirely  unaltered  state  it  possesses  as  nearly  as  possible  the 
same  index  of  refraction  as  the  rest  of  the  corpuscle.  But 
when  carbonic  acid  gas,  generated  in  a  suitable  apparatus 
(fig.  46,  I),  b'),  is  allowed  to  pass  into  the  moist  chamber  a 
tine  cloudiness  or  precipitate  occurs  around  the  nucleus,  and  the 
outline  of  this  is  brought  into  view,  whereas  if  the  carbonic 
acid  is  speedily  replaced  by  air,  which  may  be  effected 
by  disconnecting  the  tube  t  from  the  wash -bottle  b'  and  draw- 
ing air  through  it  by  the  mouth,  the  precipitate  is  re-dissolved, 
and  the  nucleus  is  again  made  to  disappear. 

It  is  of  advantage  in  performing  this  experiment  to  add  a 
trace  of  moisture  to  the  blood  before  the  observation  ;  this 
may  be  most  conveniently  done  by  breathing  two  or  three 
times  on  the  preparation  ])efore  placing  it  over  the  ring  of 
putty. 

Crystals  obtainable  from  the  colouring  matter  of  blood, 
haemoglobin  crystals. — Tlie  lia'moglobin  or  colouring  matter 
of  the  blood  may  be  obtained  in  definite  crystals,  but  the  form 
uf  the  crystals  varies  in  different  animals.  It  is  difficult  to 
induce  the  crystallisation  in  human  blood  ;  and  to  obtain  the 
crystals  readily  it  is  best  to  employ  the  blood  of  the  dog,  rat, 
guinea-pig,  or  squirrel. 

If  a  drop  of  rat's  blood  is  mixed  with  an  equal  amount  of 
distilled  water,  the  luiemoglobin  becomes  extracted  from  the 
corpuscles ;  until  this  is  done  no  crystallisation  takes  place. 
As  the  excess  of  water  begins  to  evaporate  small  needle- 
shaped  crystals  of  haemoglobin  appear,  either  singly  or  in 
bunches,  which  become  gradually  larger  until  they  may  attain 
a  very  considerable  size.  They  are  mounted  by  covering  them 
in  thick  Canada  balsam. 

Another  method  is  the  following  : — 

The  animal  is  Ijled,  and  the  blood  as  it  flows  from  tlie 
divided  vessels  is  vigorously  stirred  with  a  bundle  of  wires, 
to  remove  the  fibrin.  A  small  quantity  of  the  whipped 
blood    is   then    mixed    with    about    one-third    its    volume    of 


78  PEACTICAL  HISTOLOGY 

water,  and  a  drop  of  chloroform  ^  being  added,  the  mixture 
is  thoroughly  shaken  up  for  a  minute  or  two.  This  has  the 
effect  of  discharging  the  haemoglobin  from  the  corpuscles 
into  the  surrounding  fluid.  A  small  drop  is  now  placed  upon  a 
slide  and  left  exposed  to  the  air  for  a  few  minutes.  It  becomes 
thickened  by  evaporation  and  dried  at  the  edges,  and  crystals, 
tetrahedral  in  form  from  the  guinea-pig,  hexagonal  from  the 
squirrel,  and  rhombic  needles  from  other  animals,  may  be 
detected  in  it  with  a  low  power  of  the  microscope.  The  drop 
may  then  be  surrounded  with  thick  Canada  balsam,  covered 
and  examined  with  a  higher  power.  The  crystals  increase  in 
size  for  a  time,  and  new  ones  continue  to  be  formed. 

Haemoglobin  crystals   will  not  keep  unaltered  for  an  in- 
definite time. 

Haemiii  crystals. — The  name  '  hsemin '  has  been  given  to 
certain  very  characteristic  crystals  which  are  formed  at  the 
expense  of  the  colouring  matter  of  the  blood,  and  the  pro- 
duction of  which  is  a  trustworthy  test  of  the  presence  of 
blood,  although  yielding  no  indication  of  the  kind  of  animal 
from  which  the  blood  has  been  obtained.  To  see  them  a  very 
small  quantity  of  blood  obtained  from  the  finger  or  elsewhere 
is  smeared  upon  a  glass  slide  and  allowed  to  dry.  A  cover- 
glass  is  then  placed  over  it,  and  a  drop  of  glacial  acetic  acid 
is  applied  from  a  pipette  to  the  edge  of  the  cover-glass  and 
allowed  to  run  under  by  capillary  attraction.  The  slide  is 
then  held  by  one  end  in  the  fingers,  and  the  middle  is  gently 
warmed  over  a  small  flame,  As  soon  as  bubbles  begin  to 
appear  in  the  fluid  the  warmth  is  discontinued,  and  the  pre- 
paration is  examined  with  a  high  power.  If  no  crystals  ap- 
pear as  the  slide  cools  a  little  more  acid  is  added,  to  replace 
that  lost  by  evaporation,  and  the  slide  is  warmed  as  before, 
and  on  cooling  again  examined.  It  will  be  found  that  almost 
all  over  the  preparation  reddish-brown  short  prismatic  crystals, 
disposed  singly  or  in  groups,  will  have  made  their  appearance  ; 

1  If  nitrite  of  amyl  is  used   instead   of    chloroform,   crystals  of    viet- 
hcevwglohin  become  formed  in  place  of  lisemoglobin  (Halliburton). 


METHODS   OF  FIXING    BLOOD-CORPUSCLES  70 

most  of  them  are  very  niinute,  l)ut  they  may  be  oljtained  of 
considerable  size  by  rewarining  the  preparation  with  glacial 
acetic  acid  once  or  twice. 

The  presence  of  a  chloride  is  necessary  for  the  formation 
of  these  crystals  ;  in  the  case  of  recent  blood  the  chlorides 
which  it  naturally  contains  are  sufficient  for  the  purpose  ; 
but  if  it  were  an  old  blood-stain  which  one  had  to  deal  with, 
in  which  the  chlorides  may  have  been  washed  away,  it  would 
be  previously  requisite  to  mix  a  minute  quantity  of  common 
salt  with  the  stain  to  be  tested  in  order  to  supply  the  defi- 
ciency. 

Methods  of  fixing  and  permanently  preserving  blood- 
corpuscles. — No  reagent  is  perfectly  satisfactory  for  fixing  the 
red  blood-corpuscles,  but  osmic  acid  and  corrosive  sublimate  are 
the  best  for  the  purpose.  To  apply  the  former,  a  drop  of 
blood  is  directly  mixed  as  it  flows  from  the  pricked  finger  or 
from  the  vessels  with  an  excess  of  1  per  cent,  osmic  acid, 
which  may  contain  eosin  in  solution.  The  mixture  is 
allowed  to  stand  for  an  hour  protected  from  evaporation,  and 
some  of  it  is  then  mounted  in  dilute  glycerine. 

Another  and  on  the  whole  better  method  of  applying 
osmic  acid  is  in  the  form  of  vapour.  A  very  thin  drop  of  blood 
is  placed  upon  a  clean  cover-glass  and  instantly  laid  face 
downwards  covering  the  mouth  of  a  small  bottle  containing 
1  per  cent,  solution  of  osmic  acid.  The  vapour  of  the  acid 
fixes  the  blood-corpuscles,  and  after  the  preparation  has  re- 
mained for  a  few  minutes  in  this  position,  the  cover-glass 
with  the  layer  of  blood  is  removed  and  placed  on  a  slide  in 
a  small  drop  of  glycerine  and  water,  coloured  by  eosin.  If 
the  blood  is  too  much  clumped  together,  the  cover-glass  may 
be  gently  tapped  to  separate  the  corpuscles. 

Corrosive  sublimate  may  also  be  employed  to  fix  the  blood - 
corpuscles,  in  the  form  of  Pacini's  fluid  (mercuric  perchlo- 
ride  1  gramme,  pure  sodic  chloride  1  gramme,  distilled  water 
200  cubic  centimetres)  or  Hayem's  fluid  (see  p.  64).  The 
blood  is  mixed  with  many  times  its  volume  of  the  fluid  as 


80  PRACTICAL  HISTOLOGY 

it  flows  from  the  pricked  finger  or  from  the  vessels,  and  is 
left  for  from  15  minutes  to  several  hours  for  the  corpviscles 
to  settle.  The  fluid  is  then  decanted  off"  and  replaced  by 
rectified  spirit  containing  a  little  tincture  of  iodine,  and  after- 
wards by  96  p.c.  alcohol.  The  corpuscles  are  stained  by  dilute 
solution  of  methylene-blue  or  by  eosin,  or  by  eosin  and 
methylene-blue  in  succession. 

Blood  thus  treated  may  be  preserved  either  in  dilute 
glycerine  or  in  Canada  balsam.  The  methylene-blue  and 
eosin  stain  respectively  the  baso-phil  and  oxy-phil  granules 
of  the  leucocytes.  Eosin  also  stains  the  coloured  corpuscles. 
A  better  plan  to  stain  the  granules  of  the  leucocytes  is  the 
following  : — Make  a  preparation  of  blood  between  two  cover- 
glasses,  allow  it  to  stand  for  a  minute  or  two,  then  separate  the 
cover-glasses  and  let  the  blood  upon  them  dry  quickly  either 
at  the  ordinary  temperature  of  the  air  or  by  holding  them 
for  an  instant  over  a  flame.  If  it  is  desired  to  stain  the 
coloured  as  well  as  the  colourless  corpuscles,  the  cover-glasses 
are  either  heated  for  an  hour  or  more  to  120°  C,  or  placed  for  an 
hour  in  a  mixture  of  equal  parts  of  absolute  alcohol  and  ether, 
to  fix  the  haemoglobin  ;  if  not,  the  staining  may  be  proceeded 
with  at  once.  For  this  purpose  the  cover-glass  preparation  is 
placed  in  an  aqueous  solution  of  the  stain  which  it  is  desired  to 
employ,  e.g.  eosin  or  methyl-green,  or  any  of  those  enumerated 
under  aniline  dyes  (p.  21),  or  in  Ehrlich-Biondi  mixture,  pre- 
ferably for  some  hours,  but  the  process  may  be  accelerated  by 
warming  the  solution.  The  cover-glass  is  then  washed  with 
water,  with  acidified  spirit,  and  finally  with  absolute  alcohol, 
until  the  film  is  nearly  colourless,  when  it  is  passed  through 
xylol,  to  be  mounted  with  xylol  balsam.  The  staining  may 
sometimes  be  advantageously  effected  with  alcoholic  solutions 
of  the  dyes.' 

To  fix  the  white  blood- corpuscles  in  their  amoeboid  con- 
dition   the    following  method    may   be   recommended.     Mix 

1  See  Kanthaclt  and  Hardy,  Journ.  Physiol,  vol.  xvii.,  and  Hardy  and 
Westbrook,  Jowrn.  Physiol,  vol.  xviii. 


PREPARATION   OF   BLOOD-CORPUSCLES  HI 

newt's  blood  or  lymph  (aken  from  the  peritoneal  cavity  witli 
a  little  noi-mal  saline  solution,  cover,  and  gently  irrigate  the 
preparation  with  normal  saline  so  as  to  wash  away  most  of  the 
j)lasma  :  the  white  corpuscles  are  not  washed  away,  because  they 
tend  to  adhere  to  the  glass.  Put  the  preparation  aside  for  ten 
minutes,  by  which  time  most  of  the  white  coi'puscles  will  be 
actively  amoeboid.  Then  allow  a  jet  of  steam  from  the  spout 
of  a  kettle,  or  from  a  glass  tube  fitted  to  a  flask  of  boiling 
water,  to  play  for  one  or  two  seconds  upon  the  cover-glass, 
holding  the  preparation  close  up  to  the  spout.  The  heat 
instantly  fixes  the  corpuscles  ;  and  the  preparation  is  then 
irrigated  with  dilute  alcohol,  and  afterwards  with  dilute 
luematoxylin  or  with  one  of  the  aniline  dyes. 

After  the  corpuscles  are  stained  the  excess  of  colouring- 
solution  is  washed  away  by  a  little  dilute  alcohol  (50  per  cent.)  ; 
this,  in  its  turn,  is  replaced  by  absolute  alcohol,  and  this  by 
bergamot  oil  ;  finally  xylol  balsam  is  passed  under  the  cover- 
glass. 

A  very  simple  and  ready  method  of  obtaining  a  permanent 
stained  preparation  of  frog's  blood-corpuscles  is  the  following, 
which  is  a  modification  of  that  given  by  Stirling  : — Mix  the 
blood  directly  with  a  quantity  of  Flemming's  fluid  (p.  16).  After 
half  an  hour  decant  off  the  Flemming's  fluid  and  I'inse  with 
water,  decanting  this  off  also.  Then  add  picrocarmine  solution, 
and  allow  this  to  stand  on  the  blood-corpuscles  for  some  hours 
or  even  days.  Decant  off  the  picrocarmine,  and  mix  the  stained 
residue  of  corpuscles  with  a  little  glycerine  jelly  rendered  fluid 
by  heat.  A  drop  of  this  can  be  taken  and  mounted  at  any 
time,  for  it  will  keep  indefinitely.  The  nuclei  of  the  corpuscles 
are  stained  red,  and  the  bodies  of  the  coi'puscles  yellow.  This 
method  of  preserving  specimens  in  glycerine  jelly  is  very 
suitable  for  class  purposes,  and  may  be  applied  to  other  pre- 
pai-ations  of  isolated  elements,  such  as  macerated  epithelia, 
nerve-  and  neuroglia-cells,  muscle-fibres,  and  the  like. 


82  PEACTICAL  HISTOLOGY 


CHAPTER  II 

THE    EPITHELIAL    TISSUES 

The  epithelial  tissues  are  studied  with  regard  both  to  the 
structure  and  form  of  the  individual  elements,  and  the  rela- 
tions these  bear  to  one  another  and  to  the  membranes  they 
cover.  The  latter  class  of  observation  can  only  be  properly 
made  by  the  study  of  sections  of  the  various  organs  and  parts 
where  epithelium  is  found,  and  will  therefore  be  left  until  the 
method  of  making  these  is  explained.  The  modes  of  isolating 
and  studying  the  individual  cells  will,  however,  be  best  de- 
scribed in  this  place. 

Scaly  epithelium.     Superficial  layers. — If  a  little  of  the 
saliva  which  moistens  the  inside  of  the  cheek  be  gently  scraped 
off  with  a  small  spatula  or  with  the  finger-nail,  a  number  of 
the  superficial  cells  of  the  thick  stratified  epithelium  which  is 
here  met  with  will  be  brought  away  with  it.     The  material 
thus  obtained  is  placed  upon  a  slide  and  a  cover-glass  put  over. 
On  examining  the  preparation  numerous  flattened  scaly  epi- 
thelium cells  will  be  seen,  either  entirely  isolated  or  in  little 
patches,  the  cells  in  a  patch  being  connected  together,  with 
their  edges  overlapping.     The  cells  are  of  considerable  size, 
each  with  a  nucleus  near  its  middle,  small  in  comparison  with 
the  size  of  the  cell ;  and  the  substance  of  the  cell,  although 
clear,  yet  contains  a  number  of  scattered  granules.     Moreover, 
lines  may  often  be  seen'  running  in  various  directions  over  the 
surface  ;  these  are  for  the  most  part  due  to  creases  of  its  sub- 
stance caused  by  the  pressure  of  adjoining  cells.     Some  of  the 
cells  may  be  seen  edgeways,  and  then,  being  flattened,  will 


EPITHELIUM  83 

appear  narrow  and  linear  ;  but  on  touching  tlie  cover- glass  with 
a  bristle  their  true  form  will  be  apparent  as  they  turn  over. 

In  addition  to  such  cells  as  these  a  certain  number  of 
much  smaller  rounded  cells  may  generally  be  seen  in  the 
saliva,  which  somewhat  resemble  the  white  corpuscles  of  the 
blood,  and,  like  them,  frequently  exhibit  amoeboid  movements. 
They  are,  in  fact,  lymph-corpuscles  which  have  come  from  the 
mucous  membrane  covering  the  back  of  the  tongue  and  the 
tonsils,  which  is  very  rich  in  these  cells.  The  saliva  being  a 
watery  fluid  they  arc  swollen  out  by  it,  and  with  a  good 
microscope  it  may  be  observed  that  the  granules  in  the 
interior  of  the  corpuscles  exhibit  the  Brownian  molecular 
movement,  a  phenomenon  which,  it  will  be  remembered,  was 
exhibited  by  the  white  corpuscles  of  the  blood  as  a  first  result 
of  the  imbibition  of  water. 

There  are  several  layers  of  the  above-described  large 
flattened  epithelial  cells  in  the  epithelium  of  the  mouth. 
Below  them  are  other  cells,  smaller  and  of  a  more  spheroidal 
or  polyhedral  shape,  and  joined  to  one  another  by  fibres  which 
pass  across  fine  intercellular  spaces.  To  obtain  these  deeper 
cells  isolated,  it  is  necessary  to  macerate  a  piece  of  any  mem- 
brane which  is  covered  by  a  stratified  epithelium  in  some  fluid 
which,  while  tending  to  dissolve  the  intermediate  substance 
which  cements  the  cells  together,  may  preserve  their  natural 
form  and,  at  the  same  time,  prevent  putrefaction  from 
appearing  in  the  tissue  which  is  undergoing  maceration. 
The  best  liquid  for  this  purpose  is  a  mixture  of  alcohol  and 
water  (one  pax't  alcohol  to  two  parts  water).  The  portion  of 
tissue  must  be  small,  and  the  quantity  of  liquid  used  com- 
paratively large.  A  piece  of  the  mucous  membrane  of  the 
mouth,  pharynx,  or  gullet  of  any  mammal  may  be  used  ;  it 
will  require  at  least  two  or  three  weeks'  maceration  (for  in 
this  kind  of  epithelium  the  cells  are  very  closely  united),  and 
the  solution  should  be  changed  every  thii'd  day.  At  the  ex- 
piration of  the  time  stated  a  small  portion  of  the  epithelium 
is  scraped  oft"  with  the  point  of  a  knife,  and  placed  in  adi-op  of 

G  2 


84  PEACTICAL  HISTOLOaY 

water  upon  a  slide.  It  is  then  broken  up  as  finely  as  possible 
with  a  pair  of  mounted  needles,  a  piece  of  hair  is  cut  off  and 
placed  in  the  drop,  and  the  cover-glass  is  superposed,  after 
which  the  pieces  of  tissue  can  be  still  further  broken  up  by 
tapping  on  the  cover-glass.  By  far  the  majority  of  the  cells- 
seen  are  the  superficial  ones  already  described  ;  but  other& 
will  be  found  which  are  less  flattened  and  smaller  in  diameter, 
and  have  many  of  them  an  irregular  toothed  margin,  their 
surface  also,  as  may  be  seen  by  altering  the  fine  adjustment 
of  the  microscope,  having  on  it  linear  or  punctated  markings. 
These  are  the  so-called  cells  with  ridges  and  spines,  the  spiny 
appearance  being  caused  by  the  fibres  which  passed  from  cell 
to  cell,  and  which  have  become  broken  across  in  the  process  of 
separation.  The  cells  will  be  again  studied  in  sections  of  the 
skin,  as  well  as  in  sections  of  mucous  membranes  which  are 
covered  with  stratified  epithelium. 

Horny  layer  of  epidermis. — If  a  very  small  shred  of  the 
superficial  part  of  the  epidermis  is  taken  from  any  part,  the 
palm  of  the  hand,  for  instance,  and  examined  in  water  under 
the  microscope,  no  indications  of  cellular  structure  are  visible — ■ 
nothing  but  an  irregular  confused  mass  is  to  be  seen.  Re- 
move the  cover-glass  and  place  the  shred  of  epidermis  in  a 
drop  of  liquor  potassje.  It  will  soon  swell  up  and  become 
soft.  When  this  is  the  case  return  it  to  the  water,  and,  after 
breaking  it  up  as  finely  as  possible  with  needles,  cover  and 
examine.  Numerous  spheroidal  cells  are  now  seen  loose  in  the 
preparation,  with  a  distinct  contour,  as  if  enclosed  by  a  mem- 
brane, but  without  a  nucleus.  They  are,  in  fact,  the  scaly 
cells,  which  have  become  swollen  out  by  imbibition  of  water, 
and  at  the  same  time,  in  consequence  of  this  swelling  of  their 
surfaces,  correspondingly  diminished  in  width. 

The  same  result  may  be  obtained  with  the  cells  which 
form  the  nails ;  and  also  with  the  flattened  cells  from  the 
mucous  membrane  of  the  mouth. 

Columnar  epithelium. — This  is  most  characteristically 
seen  and  is  best  studied  as  met  with  in  the  intestinal  canal. 


COLUMNAR   EPITHELIUM  85 

It  must  be  taken  from  an  animal  (amphibian  or  mammal)  quite 
recently  killed,  as  it  rapidly  undergoes  destructive  changes  if 
left  after  death  in  contact  with  the  intestinal  contents. 
When  the  intestine  is  cut  across  at  any  part  in  a  recently 
killed  animal  the  cut  edges  curl  outwards,  and  a  little  of  the 
mucous  membrane  is  thus  exposed.  Two  very  small  portions 
may  be  snipped  oft'  this.  One  of  these  is  placed  for  a  few 
hours  in  a  few  drops  of  a  1  per  cent,  solution  of  osmic 
acid,  and  is  then  transferred  to  water,  whilst  the  other  is 
immersed  in  Flemming's  solution  diluted  with  100  times  its 
bulk  of  salt  solution,  or  in  dilute  chromic  acid  solution,  made 
by  dissolving  1  part  of  chromic  acid  in  2,000  of  salt  solution.' 
These  two  portions  may  be  put  aside  for  the  present  in  their 
respective  fluids,  whilst  a  preparation  of  the  epithelium  in  the 
fresh  condition  is  made  and  examined.  For  this  purpose  slit 
open  a  piece  of  the  intestine,  wash  away  the  mucus  and 
intestinal  contents  by  allowing  a  little  serum  or  normal  salt 
solution  to  drop  upon  the  inner  surface,  and  then,  with 
the  end  of  a  clean  scalpel,  gently  scrape  the  washed  surface, 
and  transfer  what  is  brought  away  on  the  scalpel  to  a  drop  of 
fresh  serum  upon  a  clean  glass  slide,  and  cover  the  pi'eparation, 
averting  the  pressure  of  the  cover-glass  by  means  of  a  piece  of 
hair.  On  examining  with  a  high  power  the  specimen  so  ob- 
tained numerous  columnar  epithelium  cells  will  be  seen,  some 
separate,  others  in  groups.  (It  will  be  found  advantageous 
ill  examining  the  object  to  moderate  the  illumination  by  the 
employment  of  one  of  the  smaller  holes  of  the  diaphragm  :  in 
this  way  the  some\yhat  indistinct  outlines  of  the  tissue-elements 
are  rendered  plainer,  and  any  details  of  structure  can  generally 
be  more  readily  made  out  ;  this  is  the  case,  indeed,  with  all 
unstained  preparations.)   In  the  separated  cells  the  conical  form 

'  One-third  alcohol  may  also  be  employed,  and  this  serves  to  separate  the 
cells  still  better  than  the  other  fluids,  but  their  structure  is  not  so  well  pre- 
served by  it.  The  isolated  cells  can  be  stained  with  picrocarmine  or  cannaluni 
and  preserved  in  glycerine  jelly  in  the  manner  recommended  for  the  amphibian 
blood-corpuscles  (p.  81). 


86  PEACTICAL  HISTOLOGY 

may  be  seen,  the  cell  tapering  at  one  end  to  a  point,  or  ter- 
minating in  a  rounded  or  flattened  extremity.  The  general 
substance  of  the  cell  has  a  faintly  granular  appearance  in 
the  fresh  condition,  without  a  distinct  outline,  except  at  the 
larger  end,  which  is  bounded  by  a  strongly  refracting  thickened 
margin,  in  which  a  few  faint  strise  passing  from  without 
inwards  may  with  a  high  magnifying  power  be  made  out. 
Near  the  centre  of  the  cell  is  the  clear  oval  nucleus,  bounded 
by  a  distinct  outline,  a,nd  containing  generally  one  nucleolus. 
Globules  of  fat  of  varying  size  may,  if  the  animal  were  killed 
during  digestion,  be  seen  within  the  cell ;  they  are  recognised 
by  their  strong  refractive  power. 

Of  the  groups,  some  may  occur  in  which  the  cells  are 
seen  from  above  :  the  collective  bases  will  then  present  an 
appearance  of  polygonal  areas  intersected  by  lines  of  inter- 
cellular substance  ;  in  other  groups,  where  the  cells  are  seen 
laterally,  their  arrangement  with  regard  to  one  another  will 
be  observed 

Some  cells  will  probably  be  seen  which  have  acquired  a 
peculiar  chalice-like  shape,  owing  to  the  part  of  the  cell  near 
the  free  surface  having  become  swollen  out  with  mucus,  often 
to  the  extent  of  bursting  away  and  destroying  the  free  border. 
These  are  the  so-called  'goblet  cells.' 

The  other  two  portions  of  tissue  will  not  be  ready  for 
examination  for  two  or  three  days.  A  small  piece  or  scraping 
of  the  tissue  in  the  chromic  solution  is  teased  in  a  drop  of 
distilled  water  and  covered,  with  a  small  hair  under  the  cover- 
glass  ;  and  the  small  fragments  are  further  broken  up  by  tapping 
the  cover-glass.  Staining  solution  (carmalum  or  very  dilute 
hsemalum)  is  now  allowed  to  diff'use  under  the  cover.  This 
stains  the  nuclei  and  to  some  extent  the  protoplasm  of  the  cells. 
When  the  staining  is  considered  sufficient  a  drop  of  dilute 
glycerine  is  placed  at  the  same  edge  of  the  cover-glass  to  which 
the  stain  was  applied,  and  this  also  is  allowed  to  diffuse  under 
the  cover,  which  may  subsequently  be  cemented.  Preparations 
are  made  from  the  osmic  preparation  by  gently  scraping  the 


KAKYOKINESIS  87 

epithelial  surface  and  mounting  the  product  in  dilute  glycei'ine. 
The  separation  of  the  cells  from  one  another  is  effected  by 
tapping  the  cover- glass. 

The  cells  will  be  found  to  exhibit  the  same  general 
characters  as  regards  form  and  appearance,  but  they  are 
stained  of  a  dark  grey  colour,  and  in  consequence  appear  for 
the  most  part  very  distinct.  Any  fatty  particles  which  they 
may  contain  are  coloured  intensely  black.  To  preserve  this 
prpparatioji  all  that  is  further  necessary  is  to  apply  a  little 
fixing  cement  of  some  sort  around  the  edges  of  the  cover-glass. 

Study  of  the  finer  structure  of  cells  and  nuclei.  Karyo- 
kinetic  figures. — -The  study  of  the  structure  of  cells  and  nuclei, 
both  at  rest  and  during  division,  is  in  the  first  instance  best  made 
in  the  epithelial  tissues  of  tadpoles  of  the  salamander  {Sala- 
mander maculata)  or  in  the  tail  of  the  newt  ( Triton  cristaius). 
To  obtain  salamander  tadpoles  the  female  salamanders  are  pi-o- 
cured  in  January  or  February.  They  are  viviparous,  and  at  this 
season  of  the  year  are  generally  full  of  embryo  tadpoles.  The 
latter  are  placed  in  Flemming's  or  Hermann's  solution  for  two 
days,  and  are  then  washed  for  several  hours  in  running  water, 
after  which  they  may  be  preserved  in  a  mixture  of  equal  parts 
of  glycerine,  alcohol,  and  water.  They  may  also  be  fixed  and 
hardened  in  corrosive  sublimate  solution  or  in  picric  acid  (see 
pp.  17, 18).  The  best  parts  to  take  from  the  tadpoles  are  frag- 
ments of  epidermis  from  the  end  of  the  tail  and  pieces  of  the 
gills,  or  of  the  parietal  peritoneum. 

The  following  method  is  recommended  by  Flemming. 
Thin  shreds  or  sections  of  the  fixed  and  hardened  tissue  are 
stained  for  two  days  in  a  mixture  of  equal  parts  of  alcoholic 
solution  of  saflranin  (saturated)  and  aniline- water,  then  v/ashed 
with  distilled  water,  and  differentiated  with  absolute  alcohol, 
which  may  be  acidulated  with  1  part  hydrochloric  acid  per 
1,000  ;  again  washed  in  distilled  water,  and  then  placed  in 
gentian  violet  (saturated  watery  solution)  for  one  to  three 
hours  ;  after  which  they  are  again  thoroughly  washed  with 
distilled  water.     They  are  next  rinsed  with  alcohol  until  nearly 


88  PKACTICAL  HISTOLOGY 

all  the  stain  is  washed  out,  except  from  the  nuclei,  and  the 
pieces  are  finally  transferred  to  oil  of  bergamot,  and  mounted 
at  once  in  xylol  balsam.  After  removal  from  the  gentian 
violet  and  washing  with  water  they  may  be  first  differentiated 
in  a  saturated  aqueous  solution  of  orange  G,  and  then  passed 
through  two  changes  of  absolute  alcohol  into  bergamot  oil. 

A  thick  piece  of  tissue  such  as  the  newt's  tail  must  be  cut  into 
sections  after  hardening  and  embedding  in  paraffin  and  the  sections 
fixed  on  a  sHde  (p.  37)  ;  they  may  then  be  stained  as  above.  Or 
the  hardened  tissue  may  be  stained  in  bulk  with  aqueous  solution 
of  saffranin  for  two  days,  and  after  washing  with  water  may  be  placed 
in  gum  and  cut  into  sections  by  the  freezing  method.  The  sections 
are  placed  in  water,  transferred  one  by  one  to  alcohol,  and  rinsed  in 
this  until  differentiated.  They  are  then  transferred  through  oil 
of  bergamot  to  xylol  balsam  as  before.  In  these  preparations  the 
nuclei,  especially  those  which  are  undergoing  karyokinesis,  are 
intensely  stained ;  the  nuclear  spindles  may  be  made  out  in  some 
cells,  and  in  some  the  attraction  particles  or  centrosomes  may  also 
be  detected,  especially  if  Henneguy's  method  have  been  employed 
(see  p.  21).  Simply  staining  with  very  dilute  haemalum  gives 
fairly  good  results  for  the  chromatic  nuclear  structures. 

After  fixation  in  Hermann's  fluid,  followed  by  alcohol,  and  this 
by  crude  wood  vinegar  (twelve  to  eighteen  hours),  the  chromatin 
elements  may  be  sufficiently  conspicuous  without  further  stain. 
This  method  is  recommended  by  F.  Hermann  for  the  study  of 
karyokinetic  figures  in  the  testicles  of  salamanders  killed  about 
the  end  of  July.  Thin  sections  of  the  organs  should  be  made  by 
the  paraffin  method. 

Fibres  in  epithelium  cells. — The  fibrous  structure  which  certain 
epithelial  cells  exhibit  may  be  seen  and  studied  in  the  following 
way  (Nuel)  : — Kill  a  pigeon  and  with  a  finely-drawn  glass  pipette 
immediately  inject  a  drop  or  two  of  1  per  cent,  osmic  acid  solution 
into  the  anterior  chamber  of  the  eye.  Then  cut  out  the  cornea  and 
mount  it  in  osmic  acid  with  the  posterior  surface  uppermost.  On 
this  surface  there  is  a  layer  of  flat  epithelium  cells  (endothelium  of 
Descemet's  membrane),  and  on  examining  these  cells  with  a  good 
microscope  they  are  seen  to  be  pervaded  with  fibrils  which  traverse 
the  cells  and  also  pass  across  the  intercellular  spaces  from  cell  to 
cell.  To  preserve  the  preparation  it  may  be  washed  after  being  in 
osmic  acid  for  an  hour,  and  mounted  in  dilute  glycerine. 


CILIATED   KI'lTHKUr.M  8Ji 

Intercellular  substance.  Silver  method. — To  show  the 
intercellular  substance  of  an  ei)ithc]iuni  (or  endothelium)  the 
fresh  tissue  must  be  taken,  and  after  being  rinsed  with  distilled 
water  placed  for  a  few  minutes  in  1  per  cent,  nitrate  of 
silver  solution.  On  removal  from  this  it  is  again  rinsed  with 
distilled  water  and  placed  in  tap  water  in  the  light.  In  a  few 
minutes  the  intercellular  substance  becomes  stained  brown  or 
black,  and  the  preparation  can  be  mounted  in  glycerine,  or  it 
can  be  passed  through  alcohol  and  clove-oil  into  xylol  balsam. 

Ciliated  epithelium. — For  the  present  we  may  conclude  the 
study  of  epithelium  with  the  description  of  the  modes  of  view- 
ing ciliated  epithelium,  and  of  studying  the  action  of  various 
reagents  upon  the  ciliary  motion.  The  other  more  specialised 
forms  of  epithelium,  which  are  found  in  glandular  organs  and 
elsewhere,  will  be  seen  and  studied  when  the  sevei'al  organs 
and  parts  in  which  they  occur  are  prepared. 

Ciliated  epithelium  in  its  living  state  may  be  readily 
obtained  from  the  mouth  and  gullet  of  the  recently  killed 
fi'og.  A  drop  of  aqueous  humour  should  first  be  collected 
by  passing  a  capillary  glass  tube  through  the  cornea  into  the 
antei'ior  chamber  of  the  eye  ;  the  drop  is  placed  upon  a  slide, 
and  then,  the  frog's  mouth  being  held  open  by  an  assistant,  the 
roof  is  gently  sci-aped  with  the  point  of  a  clean  scalpel,  so  as 
to  remove  the  adherent  mucus.  A  little  of  the  epithelium 
will  be  brought  away  with  this,  and  on  placing  it  in  the  fluid 
and  covering  the  preparation  (taking  the  precaution  of 
pre\iously  placing  a  hair  in  the  drop),  the  cells  may  be  sought 
for  with  a  high  power.  For  the  most  part  they  will  be 
collected  into  little  groups  of  three  or  more,  the  cilia  being  in 
acti\-e  movement  and  producing  cuiTents  in  the  liquid,  so  that 
free  particles,  blood- corpuscles,  and  other  small  objects  are 
moved  along  in  it.  But  if  the  group  is  small,  or  especially  if 
entirely  isolated  cells  are  seen,  it  will  generally  be  found  that 
the  cilia  act  upon  the  pieces  to  which  they  are  attached  like 
little  paddles,  moving  them  about  in  the  fluid.  The  cells,  it 
may  be  observed,  are  either  shortly  columnar  or  are  spheroidal  ; 


90 


PEACTICAL  HISTOLOGY 


the  nucleus  is  seldom  distinct,  because  concealed  by  the 
granular  nature  of  the  cell-protoplasm.  The  cilia  themselves 
can  best  be  seen  when  they  are  moving  languidly  or  when 
their  motion  has  altogether  stopped  ;  they  are  very  fine,  and 
spring,  a  number  together,  from  the  free  surface  of  each 
cell. 

Cilia  of  mussel. — But  by  far  the  most  convenient  object 
for  the  study  of  ciliary  motion  is  to  be  found  in  the  gill  of  the 
common  seawater  mussel  {Mytihos  edulis).  Here  the  cilia  are 
very  large,  and  their  motion  will  go  on  unimpaired  for  many 
hours.      Hence   they   are   particularly   well    suited   for   the 


Valve  of  mussel,  showing,  hr,  hr,  the  expanded  gills  or  branchise,  which,  owing- 
to  the  little  bars  of  which  they  are  composed,  present  a  striated  aspect 

ml,  mantle  ;  ni,  cut  adductor  muscle  ;  ?,  mass  of  viscera  ;  the  dark  projection  just 
above  is  the  foot 


observation  of  the  action  of  most  of  the  reagents  which  affect 
ciliary  movement. 

One  or  more  mussels  may  readily  be  procured  at  any  fish- 
monger's ;  those  only  should  be  chosen  which  remain  tightly 
closed,  for  those  with  open  valves  are  in  most  cases  already 
dead.  The  valves  may  then  be  forcibly  separated  by 
means  of  a  knife,  when  the  gills  (fig.  47  br.)  will  come  into 
view,  as  flattened  expansions  of  a  yellowish  colour,  covering 


ACTION   OF  CILIA  Ul 

a  considerable  part  of  the  shell,  inside  its  lining  membrane  ml. 
By  observing  carefully  it  may  be  noticed  that  they  have  a 
striated  aspect,  the  markings  passing  transversely  to  their 
length,  and  by  taking  up  a  small  portion  with  a  forceps  it  will 
further  be  seen  that  this  striation  is  due  to  the  fact  that  the 
gill  is  made  up  of  a  number  of  little  bars  which  are  distinct 
from  one  another  for  the  greater  part  of  their  length.  Take 
now  a  small  piece  of  the  gill,  including  three  or  four  of  the 
bars,  and  placing  it  upon  a  slide  in  a  drop  of  the  seawater 
which  the  shell  always  contains,  separate  the  bars  one  from 
another  by  means  of  needles  ;  the  preparation  may  then  be 
covered  and  observed. 

Each  of  the  bars  in  question  will  be  seen  to  be  fringed 
with  large  cilia,  which  are  set  at  an  appreciable  distance  apart 
along  nearly  its  whole  length,  but  at  the  free  extremity  of  the 
bar  are  much  more  densely  arranged.  Those  in  this  situation 
resemble  in  appearance  the  cilia  of  the  frog's  mouth,  with  the 
exception  that  they  are  very  much  longer  ;  and  like  them 
they  appear  to  spring  a  large  number  from  each  cell,  whereas 
the  others  are  stiff- looking  and  obviously  thicker,  and  are 
connected  at  their  base  each  to  a  single,  comparatively  small 
epithelium  cell.  In  spite  of  these  differences  of  appearance 
and  attachment  the  two  kinds  seem  to  be  essentially  alike 
in  nature,  for  the  mode  in  which  they  move  is  similar  and 
they  are  similarly  affected  by  reagents. 

It  will  l^e  found  that  after  a  preparation  such  as  that  just 
described  has  been  made  for  several  hours,  the  movement  will 
have  become  somewhat  languid,  and  then  the  manner  in  which 
the  individual  cilia  move  can  be  more  clearly  made  out.  The 
pi'eparation  can  be  used  also  for  the  study  of  those  agents 
which  tend  to  revive  and  stimulate  ciliary  motion,  and  it  will 
be  seen  that  it  is  precisely  the  agents  which  most  accelerate 
the  amoeboid  movements  of  the  white  blood-corpuscles  that 
have  the  most  marked  effect  upon  the  cilia  also. 

Action  of  warmth  upon  ciliary  motion. — The  same  mode 
of  applying  heat  to  a  preparation  of  cilia  is  to  be  used  as  was 


92  PEACTICAL  HISTOLOGY 

employed  for  observing  the  effect  of  warmth  upon  the  blood 
(p.  53).  It  is  well,  after  enclosing  the  preparation  with  oil 
in  the  manner  there  detailed,  to  put  it  aside  for  some  time, 
when  it  will  probably  be  found,  as  just  stated,  that  the  move- 
ment of  the  cilia  is  languid  or  altogether  arrested.  On  now 
gradually  warming  the  preparation  the  motion  becomes 
revived,  and  as  the  heat  is  raised  becomes,  pari  passu,  gradually 
faster,  until  a  point  is  reached  at  which  the  cells  are  injured 
by  the  high  temperature,  when  the  movement  slows  and  is 
arrested,  and  is  not  again  resumed.  But  if  the  experiment  be 
stopped  short  of  this  point  and  the  source  of  heat  removed,  it 
will  be  seen  that,  conversely,  as  the  temperature  of  the  stage 
falls  the  rate  of  movement  also  diminishes,  until,  when  again 
quite  cold,  the  cilia  may  again  almost  stop,  although  they 
can  be  made  to  resume  their  active  motion  on  again  applying 
warmth. 

Action  of  alkalies. — A  very  \^eak  solution  of  caustic  potash 
in  salt  solution  or  in  seawater,  similar  to  that  which  was  used 
in  investigating  the  effect  of  weak  alkalies  upon  the  blood,  may 
be  applied  to  a  preparation  of  cilia  which  have  become  some- 
what languid,  in  exactly  the  same  manner  as  in  the  case  of  the 
blood — by  allowing  a  little  of  the  fluid  to  pass  in  at  the  edge 
of  the  cover-glass  and  diffuse  itself  with  the  seawater,  so  as  to 
come  gradually  in  contact  with  the  slowly-moving  cilia.  The 
action  is  immediate  ,  the  cilia  revive  and  vigorously  lash  the 
liquid  into  which  they  project,  but  the  effort  is  soon  exhausted, 
for  the  alkaline  liquid  penetrating  the  cells  destroys  their 
vitality,  and  the  motion  of  their  cilia  stops  beyond  recovery. 
The  stimulant  action  is  not,  however,  peculiar  to  weak  alkalies, 
for  it  is  exhibited  also  by  acids  and  by  many  other  substances 
which,  applied  in  stronger  form,  would  instantly  destroy  the 
tissue,  but  when  much  diluted  tend  to  revive  and  for  a  time 
maintain  accelerated  the  ciliary  motion. 

Action  of  carbonic  acid  gas. — This  reagent  is  to  be  applied 
to  a  preparation  of  cilia  in  the  gas-chamber  in  the  manner 
directed  for  the  investigation  of  its  action  upon  the  newt's 


ACTION   OF   CILIA 


93 


blood-coipuscles  (p.  76).  Everything  being  ready,  clioose  a 
part  of  the  preparation  where  the  cilia  are  not  acting  very 
vigorously,  and  whilst  still  watching  allow  the  gas  to  pass 
over  the  preparation.  Its  immediate  action  is  seen  to  be  that 
of  a  weak  acid— that  is  to  say,  the  rate  of  movement,  if  not 
already  at  its  fastest,  becomes  accelerated — but  as  soon  as  tlie 
oxygen  of  the  air  in  the  chamber  is  entirely  displaced  by  the 
continued  stream  of  carbonic  acid  the  motion  ceases  altogether. 
As  soon  as  this  result  is  obtained  cut  off  the  stream  of  CO2 

Fig.  4S 


Apparatus  for  passing  carbonic  acid  gas  over  a  preparation  under  the 

microscope 

6,  bottle  contaimng  marble  aiul  hydrochloric  acid  :  h',  wash-bottle  ;  t,  iudia-rubber  tube 

conducting  the  gas  to  the  stage,  s 

and  reverse  the  experiment  by  blowing  air  in  at  the  side-tube, 
and  thus  displacing  the  carbonic  acid  from  the  chamber. 
The  motion  will  almost  instantly  recommence.  This  shows 
that  it  was  the  absence  of  oxygen  and  not  the  presence  of 
CO.2  which  produced  the  stoppage  of  movement ;  for  there  is, 
of  course,  an  appreciable  quantity  of  carbonic  acid  in  the  air 
which  is  thus  blown  from  the  mouth  into  the  chamber. 

Chloroform. — The    gas-chamber   i^^   again   used    for   this 


■94  PEACTICAL  HISTOLOGY 

reagent,  the  apparatus  being  arranged  in  the  way  previously 
recommended  for  blood  (p.  61).  Choosing  a  part  of  the  pre- 
paration where  the  ciliary  motion  is  vigorous,  gently  blow  a 
stream  of  the  mixture  of  air  and  chloroform  vapour  from  the 
bottle  into  the  moist  chamber.  The  cilia  become  gradually 
slower  and  eventually  stop.  Now  slip  the  indiarubber  tube 
off  the  bottle  and  gently  blow  air  through  the  chamber,  to 
displace  the  chloroform  vapour.     The  cilia  will  slowly  revive 

•  on  the  readmission  of  air,  and  will  soon  be  found  to  work 
as   vigorously  as  ever.       Like  that  with   carbonic  acid,  this 

•  experiment  can  be  repeated  a  number  of  times  with  a  like 
result,  if  the  chloroform  vapour  is  not  allowed  to  remain  too 
long  in  contact  with  the  preparation.  Other  vapours  can  be 
tested  in  the  same  way. 

Isolation  of  ciliated  cells. — To  study  the  characters  of  the 
individual  cells,  a  portion  of  membrane  which  is  covered  by 
ciliated  epithelium  is  macerated  in  some  fluid  which  softens 
and  dissolves  the  intercellular  substance,  whilst  preserving 
the  cells  themselves.  The  best  for  this  purpose  is  a  2  per  cent, 
solution  of  yellow  chromate  of  potash.  Chromic  acid  (1  in  2,000) 
or  Flemming's  solution,  diluted  100  times  with  salt  solution, 
or  ^  alcohol  may  also  be  employed.  A  large  quantity  of  the 
fluid  must  be  used,  and  the  tissue — a  piece  of  the  trachea  of 
a  rabbit  or  other  mammal,  of  the  oesophagus  of  the  frog  or  of 
the  intestinal  canal  of  a  mollusc — is  left  in  it  for  about  forty- 
eight  hours.  With  the  point  of  a  scalpel  a  little  of  the  epi- 
thelium is  then  gently  scraped  from  the  inner  surface,  and 
being  placed  in  a  drop  of  distilled  water  on  a  slide,  is  broken 
up  with  needles  as  finely  as  possible.  A  small  piece  of  hair 
is  placed  in  the  drop,  to  prevent  the  delicate  cells  from 
being  crushed  by  the  weight  of  the  cover-glass.  This  is  now 
superadded  and  the  preparation  carefully  examined  with  a 
high  power.  If  necessary  the  cover-glass  may  be  tapped  to 
separate  the  cells. 

Numerous  completely  isolated  cells  are  seen  floating  in  the 
liquid,  and  these  preserve  for  the  most  part  their  natural  form 


STKUCTURE   OF  CILIATED   CELLS  95 

and  retain  their  cilia,  although  tlie  latter  are,  of  course,  no 
longer  in  motion.  The  bright  border  through  which  the  cilia 
appeal-  to  jiass,  the  faintly  striated  cell-substance,  the  nucleus 
witli  bright  nucleolus,  and  the  truncated  ;ind  often  irregular 
fixed  extremity  of  the  cell  are,  in  most,  well  exhibited.  Besides 
these  single  cells  others  are  present  which  are  still  united  in 
groups  or  patches,  in  which,  when  viewed  from  the  surface, 
the  bases  of  the  cells  have  a  mosaic  appearance,  Moieover, 
mucus  or  '  goblet '  cells  may  here  and  there  be  met  with,  and 
other  cells  with  mucigen  granules  forming  within  them. 

These  are  destitute  of  cilia  ;  they  lie,  in  the  natural  state, 
between  the  ciliated  cells,  and  they  may  occasionally  be  seen 
in  situ  in  some  of  the  small  groups  of  cells  which  have  remained 
attached  to  one  another. 

If  it  be  wished  to  permanently  preserve  such  a  preparation 
as  that  now  under  description,  it  is  necessary  first  to  stain  the 
cells  somewhat  and  then  to  substitute  glycerine  for  the  stain- 
ing Huid.  Either  carmalum  or  dilute  lia^malum  or  1  per 
cent,  osmic  acid  solution  may  be  used  for  staining  the  cells. 
The  two  former  mainly  colour  the  nuclei  very  intensely,  the 
last  gives  a  uniform  grey  tint  to  the  cells.  The  colouring 
fluid  is  applied  in  the  following  manner  : — A  drop  is  brought 
in  contact  with  one  edge  of  the  cover-glass.  AYhen  the 
staining  fluid  has  diff"used  into  the  water  in  which  the  pi-epara- 
tion  was  made,  the  preparation  is  left  until  the  cells  appear 
sufficiently  coloured,  a  little  more  fluid  being  occasionally  added 
if  there  seems  any  danger  of  the  specimen  becoming  dry. 
With  a  solution  of  haniialum,  even  though  very  dilute,  a  few 
minutes  suthce  ;  the  osmic  acid  solution  should  be  allowed  to 
remain  an  hour  in  contact  with  the  cells.  The  staining  fluid 
is  replaced  by  a  small  drop  of  glycei-ine  and  water,  which  is 
applied  at  one  border  of  the  cover-glass  and  gradually  takes 
the  place  of  the  water  as  this  evaporates  at  the  edges.  The 
cover-glass  may  then  be  fixed. ^ 

1  For  class  purposes  the  method   recommendecT  on  p    81  will  be  fouud 
valuable. 


96  PE ACTIO AL  HISTOLOGY 


CHAPTER  III 

CONNECTIVE     TISSUE 

In  the  areolar  tissue  and  in  connective  tissue  generally  there 
are  several  parts  which  present  themselves  for  study  ;  and  in 
order  to  observe  each  to  the  greatest  advantage  different 
modes  of  preparation  are  for  the  most  part  requisite. 

The  fibres  of  areolar  tissue.  Ranvier's  demidesiccation 
method. — For  the  observation  of  the  fibrous  elements,  simply, 
without  special  regard  to  their  arrangement  or  relation  to  the 
other  elements,  all  that  is  necessary  is  to  place  a  small  portion 
of  areolar  tissue,  taken  from  any  part,  on  the  centre  of  a  glass 
slide,  just  moistened  with  the  breath,  and  with  clean,  sharp 
needles  separate  it  as  finely  as  possible  into  filamentous  shreds. 
Then,  without  allowing  the  preparation  to  become  dry,  take  a 
cover-glass  on  which  a  drop  of  salt  solution  has  previously 
been  placed  and  invert  this  over  the  tissue.  The  object  of 
using  but  very  little  fluid  to  prepare  the  tissue  in  is  to  prevent 
the  filaments  from  running  together  and  becoming  entangled 
when  released  from  the  needles. 

In  a  preparation  so  made  nothing  is  as  a  rule  apparent 
save  the  wavy  bundles  of  connective  tissue  fibrils,  these  when 
much  developed  obscuring  the  elastic  fibres  and  corpuscles  of 
the  tissue.  But  if  a  second  preparation  be  made  in  precisely 
the  same  way,  except  that,  in  place  of  salt  solution 
simply,  salt  solution  containing  1  part  of  acetic  acid  in  200 
is  placed  upon  the  cover-glass,  and  if  then  the  object  is 
imraediately  examined  with  a  high  power,  it  is  seen  that  the 
fibrils  which  compose  the  bundles  gradually  become  indistinct. 


AKEOLAR  TISSUE  1)7 

whilst  the  bundles  are  soon  iniioh  .swollen,  except,  it  may  be, 
at  intervals  here  and  there.  At  the  same  time  certain  other 
fibres,  almost  equally  line  but  more  sharply  defined  than  tli<( 
white  fibrils,  and  always  running  singly,  never  in  bundles, 
come  into  view.  These  are  the  elastic  fibres.  If  one  of  them 
be  followed  for  a  short  distance  it  will  probably  be  seen  that 
it  sooner  or  later  gives  off  a  branch  which  unites  it  with  a 
neighbouring  fibre,  whereas  the  white  fibrils  never  show  any 
disposition  to  branch  or  unite  with  one  another,  but  those  in 
each  bundle  maintain  from  end  to  end  a  perfectly  parallel 
course.  The  elastic  nature  of  the  filaments  which  are  brought 
into  view  by  acetic  acid  is  shown,  in  such  a  preparation  as  we 
are  describing,  by  the  fact  that  wherever  in  the  process  of  teas- 
ing the  tissue  they  have  become  broken  across,  the  fibres  have, 
by  the  recoil  from  the  stretching  to  which  they  were  submitted 
before  the  ruptui'e  occurred,  been  thrown  into  bold  curves, 
especially  marked  near  the  broken  extremities,  which  are  often 
recurved.  That  this  curved  or  coiled  appearance  of  the  elastic 
fibres,  although  highly  characteristic,  and  always  observable 
when  the  tissue  is  thus  prepared,  is,  however,  not  a  natural 
one,  is  shown  by  the  fact  that,  as  will  immediately  be  described, 
when  precautious  are  taken  to  preserve  as  much  as  possible 
the  normal  arrangement  of  the  tissue-elements  the  elastic 
fibres  are  seen  to  pursue  a  rectilinear  course. 

The  cells  of  areolar  tissue. — To  demonstrate  the  cells  or 
connective  tissue  corpuscles  the  preparation  is  made  more 
methodically.  A  film  as  thin  as  possible  must  be  obtained 
for  observation,  so  as  to  avoid  the  necessity  of  tearing  the 
tissue.  Such  films  are  naturally  present  in  the  areolar  tissue 
of  most  parts,  and  may  be  seen  when  the  organs  which  it 
connects  are  gently  drawn  asunder  from  one  another,  as,  for 
instance,  when  the  skin  is  raised  and  reflected  from  the 
subjacent  fasciae  and  muscles.  The  most  convenient  soui-ce  of 
such  a  delicate  film  is  to  be  found  in  the  exquisitely  thin  and 
transparent  tissue  which  invests  and  lies  between  the  muscles 
of  the  fore-limb  of  the  rabbit  and  guinea-pig.     The  tissue  in 

H 


98  PKACTICAL  HISTOLOGY 

this  situation,  especially  if  taken  from  a  young  animal,  is 
devoid  of  fat  and  not  so  completely  overridden  by  the  bundles 
of  white  fibrils  but  that  the  elastic  fibres  and  the  connective 
tissue  corpuscles  can  be  made  out  even  without  the  addition 
of  reagents.     The  mode  of  preparation  is  as  follows  : — 

The  animal  having  been  killed  by  bleeding,  the  skin  is 
snipped  through  around  the  upper  part  of  the  fore-limb  and 
is  then  forcibly  reflected  from  the  limb.  In  this  operation 
care  must  be  taken  to  avoid  besprinkling  the  subjacent  parts 
with  the  cut  hairs  of  the  animal.  A  piece  of  the  tissue 
over  or  between  the  muscles  is  then  seized  with  forceps  and 
snipped  off"  with  sharp,  fine  scissors.  The  snipped-ofi"  tissue 
shrinks  immediately  around  the  end  of  the  forceps  and  appears 
very  unsuited  for  microscopical  examination.  But  place  it  on 
a  clean  slide,  without  the  addition  of  any  fluid,  and  with  a 
pair  of  mounted  needles  endeavour,  by  drawing  out  first  this 
corner  and  then  that,  to  again  reduce  the  gelatinous-looking 
piece  to  the  condition  of  a  thin  film,  and  it  will  be  found  that 
this  can  be  effected  without  much  difficulty,  for  when  not 
floated  up  by  fluid  the  thin  edges  of  the  film  tend  to  stick  to 
the  glass,  and  cease  to  shrink  away  from  the  position  to  which 
they  are  drawn  by  the  needles.  At  the  same  time,  whilst  it 
is  important  not  to  add  fluid  to  that  which  naturally  moistens 
the  piece  of  tissue,  it  is  equally  important  never,  during  the 
whole  process  of  stretching,  to  let  the  film  become  actually 
desiccated,  for  this  would  altogether  ruin  the  tissue  for  micro- 
scopic purposes.  The  best  way  to  prevent  such  a  result  is  to 
breathe  now  and  then  on  the  object  whilst  it  is  being  prepared  ; 
by  so  doing  needless  haste  will  be  avoided  and  more  time  and 
pains  can  be  taken  for  the  complete  display  of  the  film.  This 
being  effected,  a  cover-glass  (which  should  have  been  previously 
cleansed  and  placed  in  readiness,  with  a  drop  of  salt  solution 
upon  it)  is  taken,  and  quickly  superposed  over  the  film  of 
tissue,  which  is  thus  prevented  from  shrinking  up  again  into  a 
shapeless  mass.  The  fibres,  botb  the  white  (in  wavy  bundles 
of  various  sizes)  and  the  elastic,  and  the  corpuscles  may  now 


AlvEOLAK   TISSUE  1)«) 

be  carefully  observed,  at  first  with  the  usual  high  power  and 
afterwards  with  the  highest  ol^tainable,  and  some  of  the  cor- 
puscles should  be  sketchefl.  ^Moreover,  search  may  be  made  for 
lymph-corpuscles,  a  very  few  of  which  are  generally  to  bo 
found  in  the  connective  tissue  ;  they  are  readily  distinguished 
from  the  fixed  corpuscles  of  the  tissue  by  their  smaller  size — 
small,  obscure,  and  generally  multitid  nuclei  and  especially 
their  amceboid  movements,  of  which  it  is  probable  no  trace 
will  be  apparent  even  to  the  most  assiduous  observation  in 
the  connective  tissue  cells  proper. 

Although  both  corpuscles  and  elastic  fibres  may  be  seen 
in  a  preparation  of  this  kind  made  with  an  indifferent  fluid, 
they  are  better  seen  if  the  white  fibres  are  acted  upon  by  acetic 
acid,  and  still  better  if  this  action  is  combined  with  that  of 
h;ematoxylin,  so  that  the  corpuscles  are  brought  more  pro- 
minently into  view.  Moreover,  the  preparation  admits  of 
being  permanently  preserved  in  glycerine  after  such  a  method 
of  treatment.  Up  to  a  certain  stage  the  procedure  is  the  same 
as  that  above  described,  but  instead  of  placing  salt  solution 
upon  the  cover-glass  before  inverting  it  over  the  film,  a  solu- 
tion of  acetic  acid  (1  per  cent.)  is  used,  which  tends  to  swell 
u[)  the  white  fibres  and  thus  brings  the  cells  and  elastic  fibres 
clearly  into  view.  The  cells  can  then  be  stained  by  runnin'>- 
h;\}malum  solution  under  the  cover-glass.  Very  good  results 
may  also  be  got  by  applying  carmalum  (nothsemalum)  directly 
to  a  film,  without  the  previous  action  of  acetic  acid. 

The  connective  tissue  corpuscles  can  probably  be  made 
out  at  once  in  the  thinner  parts  of  the  prepai'ation,  with  their 
clear  oval  nuclei  and  the  flattened  irregular  area  occupied  by 
their  cell-substance.  In  a  few  minutes  their  nuclei  will  be 
tinted  by  the  stain,  and  will  then  show  up  much  more 
prominently  ;  but  to  get  the  cell-bodies  sufliciently  coloured 
it  will  be  necessary  to  leave  the  staining  solution  half  an  hour 
or  more  in  contact  with  the  preparation.  Meanwhile,  to 
obviate  the  effects  of  evaporation,  a  considerable  drop  of  the 
colouring  fluid  should  be  placed  on  either  side  in  contact  with 

u  -2 


100  PRACTICAL  HISTOLOGY 

the  edges  of  the  cover-glass.  The  excess  of  fluid,  moreover, 
has  a  tendency  to  raise  the  latter  slightly  from  the  film  of 
tissue,  and  in  this  way  a  more  ready  access  of  fresh  colouring- 
fluid  is  permitted.  When  it  is  found  on  examination  that  the 
corpuscles  are  properly  stained,  the  solution  may  be  drawn  ofi" 
by  a  slip  of  filter  paper  applied  to  the  edge  of  the  cover- 
glass  on  one  side,  whilst  to  the  other  water  is  applied  and 
frequently  renewed,  the  excess  of  stain  being  in  this  manner 
rinsed  away.  A  drop  of  glycerine  and  water  coloured  by 
magenta  is  now  drawn  under  the  cover-glass ;  the  magenta 
serves  to  stain  the  elastic  fibres,  and  the  glycerine  permanently 
preserves  the  preparation  ;  the  cover-glass  may  then  be  at 
once  cemented.  Picrocarmine  may  also  be  employed  as  a 
stain,  but,  as  it  only  acts  slowly,  it  is  necessary  to  keep  the 
preparation  in  a  moist  chamber  for  some  hours  to  prevent  the 
evaporation  of  the  water. 

In  specimens  treated  with  acid  there  maybe  observed  a  con- 
stricting ring  at  intervals  along  the  course  of  some  of  the  connective 
tissue  bundles,  an  appearance  which  has  long  been  familiar  to 
histologists.  The  nature  of  this  constricting  ring  is  not  yet  clearly 
determined,  some  supposing  it,  from  its  resistance  to  the  action  of 
acids,  to  be  of  the  nature  of  elastic  tissue,  others  that  the  appear- 
ance is  caused  by  the  process  of  a  corpuscle  enwrapping  the  bundle. 
As  shown  by  Ranvier,the  constricting  ring  is,  like  the  cells,  tinted 
red  by  picrocarmine,  whilst  the  elastic  fibres  are  coloured  yellow. 

Preparation  of  denser  areolar  tissue. — In  most  of  the 
larger  animals  {e.g.  the  dog)  the  connective  tissue  is,  in  the 
adult,  so  densely  pervaded  by  bundles  of  white  fibres  as  to 
render  it  impossible  to  obtain  a  film  delicate  enough  for  easy 
observation  without  tearing  the  texture  with  needles,  and 
thereby  distorting  the  cellular  elements.  Or  it  may  be  desired, 
even  in  those  animals  in  which  such  delicate  tissue  as  that 
the  preparation  of  which  has  just  been  described  is  found,  to 
obtain  a  specimen  from  a  part  where  the  connective  tissue 
is  not  naturally  extended  in  so  advantageous  a  manner  for 


AREOLAR  TISSUE  101 

preparation  and  observation.     In  these  cases    tlu;   following 
nu'thod  may  be  employed  with  advantage  : — 

In  a  recently  killed  dog  a  flap  of  skin  is  dissected  back, 
and  a  hypodermic  syringe  (fig.  49),  provided  with  a  fine 
cannula,  having  been  previously  filled  with  salt  solution,  the 
point  of  the  cannula  is  inserted  underneath  the  layer  of  con- 
nective tissue  which  is  most  superficial  on  the  reflected 
portion  of  skin,  and  a  little  of  the  fluid  is  forced  out.  This 
docs  not  immediately  diffuse  itself  uniformly  through  the 
loose  areolar  tissue,  but  remains  for  a  short  while  at  the  same 
place,  forming  a  little  bulla  of  liquid  bounded  and  covered 
in  by  a  film  of  tissue,  the  thickness  of  which  depends  upon 

Fig.  49 


=^si 


Syringe  for  interstitial  injections  ;  c,  the  cannula.  Tlie  sj-ringe  is  glass  with 
ebonite  fittings ;  the  cannula  is  a  fine  perforated  steel  needle  set  in 
ebonite.     Natural  size. 

the  depth  to  which  the  cannula  was  inserted.  If  it  does  not 
appear  thin  enough  a  second  attempt  should  be  made  at 
another  spot.  Then,  before  the  bulla  has  time  to  subside — 
that  is  to  say,  before  the  fluid  has  time  to  diffuse  itself  through 
the  meshes  of  the  tissue — snip  off"  the  whole  projection  with  a 
single  cut  of  a  pair  of  scissors,  which  for  this  purpose  should 
be  particularly  sharp  and  clean,  and  transfer  the  snipped-off 
portion  to  a  clean  slide.  Here  it  may  either  be  at  once 
,  covered  in  salt  solution  and  examined  without  reagents,  or 
may  be  treated  with  acetic  acid,  hsematoxylin,  itc,  as  in  the 
mode  of  preparation  just  described. 

A  modification  of  the  above  method  consists  in  injecting 
into  the  tissue  a  solution  of  gelatine  instead  of  salt  solution. 


102  PKACTICAL  HISTOLOGY 

The  gelatine  solution  is  made  by  taking  some  clear  French  gela- 
tine, allowing  it  to  soak  for  an  hour  or  two  in  water,  and  then, 
after  pouring  off  all  the  excess  of  that  fluid,  placing  the  soaked 
gelatine  in  a  beaker  over  a  water-bath  until  it  is  entirely 
melted  in  the  water  which  it  has  imbibed.  The  syringe  is 
then  warmed  by  immersing  it  for  a  minute  or  two  in  warm 
water,  and  is  filled  with  the  gelatine  solution,  and  a  little  of 
this  is  injected  into  the  subcutaneous  connective  tissue,  so  as 
to  produce  a  bulla  like  that  made  by  the  salt  solution.  In 
cold  weather  the  gelatine  will  set  almost  immediately  ;  in 
warm  weather  the  process  may  be  accelerated  by  surrounding 
the  bulla  with  small  pieces  of  ice. 

When  the  gelatine  is  quite  firm,  sections  of  it  are  made 
with  a  razor.     As  they  are  cut  they  are  placed  in  salt  solution. 

Before  mounting  them  it  is  well  to  stain  the  specimens, 
and  this  may  advantageously  be  done  by  aqueous  solution 
of  magenta.  This  colours  the  elastic  fibres  strongly,  the 
corpuscles  distinctly,  and  the  bundles  of  white  fibres  slightly, 
while  the  gelatine  which  was  injected,  and  of  course  occu- 
pies all  the  interstices,  is  hardly  stained  at  all.  The  time 
of  immersing  the  sections  varies  of  course  with  the  strength 
of  the  fluid,  but  this  should  not  be  too  highly  coloured,  and 
it  can  be  then  seen  without  much  difficulty  when  the  sections 
are  sufficiently  stained.  They  are  subsequently  placed  in 
water  for  a  minute  or  two  to  remove  the  excess  of  magenta 
prior  to  transferring  them  to  a  slide.  A  drop  of  glycerine 
is  now  added  and  the  cover-glass  laid  on,  after  which  the 
slide  is  gently  warmed  over  a  small  flame  or  otherwise  until 
the  gelatine  in  the  sections  just  melts,  so  as  to  allow  the 
cover-glass,  which  was  probably  tilted  up  somewhat  owing  to 
the  thickness  of  the  sections,  to  settle  down.  The  specimen 
may  then  be  examined,  and  if  satisfactory  may  be  preserved, 
the  preparation  being  completed  by  fixing  the  edges  of  the 
cover-glass  with  gold  size. 

By  the  modes  of  preparing  connective  tissue  already  described, 
most  of  which  we  owe  to  Eanvier,  the  bmidles  of  white  fibres,  the 


AREOLAR  TISSUE  10;> 

elastic  fibres,  and  the  corpuscles  are  brought  imder  observation,  and 
it  would  seem  at  first  sight  that  these  of  tliemselves  entirely 
constitute  the  tissue.  But  in  considering  the  nature  of  the  fihus 
obtained — that  they  are,  namely,  continuous  over  a  greater  or  less 
area— it  is  clear  that  the  presence  of  fibres  and  cells  is  not  alone 
suthcient  to  account  for  the  laminaj  which  are  spread  upon  the 
shde.  And,  indeed,  by  closely  observing  the  preparation  it  will  be 
apparent  that  there  is  pellucid  substance  uniting  everything  together, 
thi-ough  which  the  fibres  run,  and  in  which  the  corpuscles  lie  em- 
bedded. There  is,  it  is  true,  a  difficulty  in  making  this  out  in  most 
parts,  in  consequehce  of  its  extreme  clearness,  and  the  fact  that 
its  refractive  index  is  little  different  from  that  of  the  watery  fluid 
the  tissue  is  examined  in ;  moreover,  in  the  logwood  preparations 
the  intermediate  substance  remains  entirely  unstained.  Never- 
theless, towards  the  edge  of  the  preparation,  where  a  comparison 
can  the  better  be  made  with  the  surrounding  fluid,  the  fact  that 
such  a  clear  inter-mediate  substance  does  really  exist  will  be 
sufficiently  evident ;  and  the  more  so  if  the  cover-glass  be  slightly 
moved,  or  one  edge  be  gently  ^Dressed  down  with  a  needle.  But 
we  possess  in  the  silver  method  of  Eecklinghausen  a  ready  means 
of  demonstrating  its  existence  in  an  obvious  manner ;  for  the 
gromid- substance  (or  intercellular  substance)  of  the  connective 
tisue,  and,  indeed,  of  almost  every  other  tissue  in  the  body, 
possesses  the  distinctive  propert,y  of  reducing  the  salts  of  silver 
under  the  action  of  light,  so  that  the  metal  is  deposited  in  it. 
The  effect  of  this  deposition  is  to  stain  the  ground-substance  of 
a  colour  varying  with  the  intensity  of  the  light  employed  and  with 
other  conditions  from  a  light  brown  to  a  brownish  black.  The 
fibrous  elements  participate  for  the  most  part  in  this  staining,  and 
are  frequently,  especially  when  the  preparation  has  been,  as  is 
usual  with  silver  preparations,  mounted  in  gl3'cerine,  indistinguish- 
able from  the  gromid- substance  through  which  they  course,  and 
which  also  miites  the  white  fibres  into  the  bmidles  which  they  form. 
The  cellular  elements,  on  the  contrary,  remain  absolutely  imstained, 
and,  moreover,  after  the  action  of  the  silver  salt  are  no  longer 
affected  by  those  stainmg  fluids  which  otherwise  have  a  particular 
affinity  for  them  ;  it  is  therefore  no  longer  possible  to  bring  them 
into  view.  "Wherever,  then,  a  cell  is  situated,  there  appears  after 
the  reduction  of  the  silver  nothing  but  a  white  patch  upon  or  in 
the  brown  ground ;  and  if,  as  is  not  unfrequently  the  case,  several 
flattened  cells  may  have  occurred  together  with  their  edges  in  jux- 


104  PEACTICAL  HISTOLOGY 

taposition,  the  group  appears  as  a  larger  white  patch  mtersectedby 
dark  lines,  these  representing  a  small  amount  of  intercellular  sub- 
stance between  the  individual  cells.  The  appearance  is  similar  to 
what  is  observed  in  an  epithelial  tissue  after  the  silver  treatment, 
for  in  this  the  intercellular  substance  is  always  very  small  in 
amount.  Such  an  arrangement  of  connective  tissue  cells  is  on  this 
accotmt  designated  '  epithelioid.'  The  white  patches,  then,  in  the 
silvered  preparation  of  connective  tissue  represent  either  depres- 
sions on  the  surface  of,  or  acttial  cavities  within,  the  matrix  or 
ground-substance,  containing  cells,  which  themselves  are  not  visible, 
so  that  the  white  patches  are  tevmed  the  cell-spaces  ov  (recalling 
the  analogous  case  of  bone)  the  lacunce  of  the  connective  tissue. 

It  is  the  more  appropriate  to  give  them  a  special  designation 
because,  as  may  be  made  out  by  a  careful  comparison  of  specimens 
of  connective  tissue  from  the  same  part,  some  prepared  with  chloride 
of  gold,  to  show  the  cells,  others  with  silver,  to  show  the  ceU-spaces, 
the  cell-spaces  are  in  many  cases  distinctly  larger  than  the  cells ; 
they  are  not  necessarily  therefore,  as  has  sometimes  been  supposed, 
and  as  is  no  doubt  the  case  with  the  clear  part  of  a  silvered  epithe- 
lium, merely  the  cells  left  white.  The  difference  in  the  relative 
size  of  the  cell-spaces  and  the  contained  cells  obtains  no  doubt  more 
frequently,  or  at  least  can  be  more  readily  made  evident,  in  the 
firmer  varieties  of  connective  tissue,  where  the  groiuid-substance 
is  everywhere  pervaded  with  fibrous  bundles,  and  has  in  conse- 
quence lost  its  soft  and  yielding  nature,  which  otherwise  permits  it 
to  adapt  itself  more  readilj^  to  the  shape  of  the  cells. 

Thus  much  having  been  said  in  order  to  explain  the  appear- 
ances produced  by  the  silver  method  of  treatment,  the  best 
mode  of  applying  it  to  ordinary  connective  tissue,  such,  for 
instance,  as  the  subcutaneous,  may  now  be  described. 

Application  of  silver  method  to  areolar  tissue :  cell-spaces 
and  intercellular  substance. — The  skin  of  a  recently  killed 
rabbit  or  guinea-pig  ^  having  been  stripped  off  one  of  the  limbs, 

'  These  animals  are  selected  because  there  is  likely  to  be  less  fat  in  the 
subcutaneous  tissue  than  in  that  of  the  cat  or  dog  or  other  animals  commonly- 
used  in  the  laboratory.  It  is  important  to  remember  that  any  tissue  which  is 
to  be  submitted  to  the  silver  niethod  must  be  fresh  and  unacted  upon  pre- 
■  viously  by  any  reagent  whatever ;  moreover  any  blood  on  the  part  must  be 
rinsed  away  with  distilled  water. 


SILVER  METHOD  105 

this  is  disarticulated  at  tlie  proximal  joint,  aiid  is  rinsed  for  a 
second  or  two  in  a  beaker  of  distilled  water,  in  order  to  wash 
away  any  l)lood  or  lymph  which  mi^ht  happen  to  be  on  the 
surface,  and  which  would  cause  a  granular  precipitate  with 
the  nitrate  of  silver  solution.     The  latter,  a  solution  of  1  part 
of  the  salt  to  100  of  distilled  watei-,   is  then  either  poured 
over  the  surface  or  dropped  on  it  from  a  pipette.     After  two 
or  three  minutes  the  silver  solution  is  quickly  washed  off  by 
a  stream    of   distilled    water,    and  the  limb  is  then  at  once 
placed  in  a  beaker  of  spirit,  and  exposed  to  direct  sunlight, 
or,  failing  this,  to  bright  diffused  daylight.     In  a  few  minutes 
in  the  sunlight,  and  after  a  longer  time  in  diffused  daylight, 
the  silvered  surface  will  have  acquired  a  uniform  brownish 
tinge  to  the  naked  eye.     When  the  colour  is  strongly  marked 
it  is  as  well  to  remove  the  beaker  from  the  light,  lest  the 
preparation  become  too  darkly  stained.     The  limb  should  be 
allowed  to  remain  in  spirit  during  twenty-four  hours  ;  at  the 
expiration  of  this  time  it  is  placed  in  a  dish,  and,  by  the  aid 
of  fine  forceps  and  scissors,  pieces  of  the  superficial  stained 
layer  are  dissected  off  under  spirit.     In  doing  this  care  must 
be  taken  not  to  drag  at  all  upon  the  membrane  which  is  thus 
removed,  so  as  to  throw  it  into  creases.     Or  thin  slices  may 
be  cut  with  a  razor  from  the  surface  browned  by  the  silver. 
The  pieces  are  then  transferred  to  oil  of  cloves  on  a  clean 
glass  slide  ;  and  most  of  the  superfluous  oil  of  cloves  having 
been  wiped   away,  a    cover-glass,  on  which  a  drop  of   xylol 
balsam    has  been  placed,  is   inverted   over   the  preparation. 
Before  putting  on  the  cover-glass  it  is  well  to  examine  the 
object  under  a  low  power,  in  order  to  make  sure  of  the  absence 
<^f  folds  and  creases  or  specks  of  dust  upon  it  :  if  any  such 
be   seen    they   must   be    carefully    removed   with   a   needle. 
Indeed,  it  may  be  recommended  as  a  golden  rule  in  making 
histological  preparations  never  to  put  the  cover-glass  in  its 
place  until  a  glance  at  the  object  under  a  low  power  of  the 
microscope  has  certified  the  absence  of   any  marked  imper- 
fection :  if  this  be  attended  to  the  time  will  often  be  saved 


106  PEACTICAL  HISTOLOGY 

which    would    otherwise    be    spent    in    mounting   worthless 
specimens. 

A  simpler  method  of  showing  the  ground- substance  and 
cell-spaces  of  the  subcutaneous  tissue  consists  in  making  a 
film  by  the  method  of  demidesiccation  as  detailed  on  p.  98,  and 
placing  a  drop  of  1  per  cent,  nitrate  of  silver  solution  upon 
the  middle  of  the  film.  After  a  minute  or  two  the  silver  nitrate 
is  rinsed  oS  with  distilled  water  and  the  preparation  is  placed 
for  a  few  minutes  in  sunlight.  The  water  may  then  be  re- 
moved by  alcohol,  this  replaced  by  clove-oil  and  the  prepara 
tion  finally  mounted  in  xylol  balsam. 

The  connective  tissue  which  covers  the  tendons  of  the 
superficial  flexor  digitorum  of  the  ox's  foot,  as  they  run 
through  sheaths  formed  by  the  tendons  of  the  deep  flexor, 
is  more  easily  prepared  by  the  silver  method  than  the  looser 
kinds  such  as  the  subcutaneous  tissue.  A  piece  of  such 
a  tendon  is  taken,  rinsed  in  distilled  water  to  remove  the 
synovial  fluid  which  covers  it,  and  treated  with  silver  in  the 
way  described  on  the  preceding  page  ;  and  is  then,  after  wash- 
ing, placed  in  the  light  in  spirit.  It  soon  becomes  brown,  when 
it  may  be  removed  from  the  light ;  and  after  remaining  twenty- 
four  hours  in  the  spirit  it  is  easy,  with  a  sharp  knife  or  razor 
wetted  with  spirit,  to  obtain  a  thin  surface  section.  This, 
after  being  immersed  for  a  minute  or  two  in  clove- oil  to  get 
rid  of  the  spirit,  is  mounted  in  xylol  balsam  with  the  browned 
surface  uppermost.  It  should  present,  if  successful,  an  ex- 
tremely characteristic  and  beautiful  image  of  white  branched 
cell-spaces,  single  or  in  groups,  upon  a  brown  ground. 

In  both  the  preparations  last  described  it  is  possible  to 
show  the  nuclei  of  the  corpuscles  which  lie  in  the  cell-spaces 

by  subsequent  staining  with  logwood.     Sometimes  they  are 

visible  even  without  this  treatment. 

Elastic  network  of  areolar  tissue. — The  proportion  of 

elastic  fibres  varies  considerably,  according  to  the  part  from 

which  the    connective   tissue   under   examination   is    taken. 

Some  serous   membranes    contain  a  large  number  of  elastic 


EJ-ASTIC    I'limES  107 

fibres  ;  and  since  they  are  readily  spread  out  in  their  natui-al 
condition  the  network  which  these  fibres  form  by  their  branch- 
iiiji;s  and  conjunctions  is  easily  made  evident.  One  of  the  best 
objects  for  this  purpose  is  to  be  found  in  the  rabbit's  mesocolon. 
Apiece  of  this,  moistened  witli  a  little  salt  solution,  may  be 
spread  out  as  flat  as  possible  upon  a  slide,  and  a  drop  of  dilute 
magenta  solution  having  l)eeu  placed  on  a  cover-glass,  this 
is  inverted  over  the  tissue.  The  elastic  network  becomes 
stained  by  the  dye,  whilst  the  white  fibres  remain  almost 
unstained.  The  preparation  may  be  made  permanent  by 
putting,  as  in  former  preparations,  a  drop  of  dilute  glycerine 
at  the  edge  of  the  cover-glass,  and  after  this  has  had  time  to 
diffuse  itself  cementing  the  edges  with  gold  size. 

Elastic  tissue. — The  elastic  ligaments  are  connective  tissue 
structures  in  which  the  elastic  elements  of  the  tissue  greatly 
preponderate.  There  is  always  a  quantity  of  ordinary  areolar 
tissue  amongst  the  fibres,  but  not  sufficient  to  obscure  them, 
especially  as  they  are  generally  of  larger  size  as  well  as  in 
greater  number  than  elsewhere.  It  is  sufficient,  in  order  to 
see  them,  simply  to  teaze  out  a  portion  of  the  ligamenta 
subliava  of  the  vertebrcC  or  of  the  ligamentum  nuchas  of  the 
ox  in  water  or  salt  solution.  If  it  be  desired  to  keep  the 
preparation  it  can  be  mounted  in  glycerine  or  glycerine-jelly. 

Section  of  elastic  fibres. — To  observe  the  shape  of  the  fibres 
a  transverse  section  may  be  made  of  a  piece  of  ligamentum 
nuchse  of  the  ox,  in  which  the  fibres  are  extremely  large. 

In  order  to  obtain  the  requisite  firmness  for  cutting,  place 
a  small  piece  of  the  ligament  in  a  quantity  of  2  per  cent, 
solution  of  bichromate  of  potash  for  fourteen  days  ;  then  in 
water  for  two  or  three  hours  ;  then  transfer  to  gum.  When 
soaked  with  this,  sections  across  the  direction  of  the  fibres  may 
be  made  with  the  freezing  microtome.  The  sections  are  to  be 
placed  in  water  to  remove  the  gum,  and  mounted  in  glycerine, 
either  with  or  without  staining  ^dth  magenta  or  hiiematoxylin. 

Fibrous  or  tendinous  tissue.  This  may  be  examined  by 
separating  a  small  shred  from  a  tendon  or  ligament,  and  teasing 


108  PEACTICAL  HISTOLOGY 

it  out  as  finely  as  possible  into  its  constituent  bundles.  The 
operation  is  conducted  with  the  aid  of  needles  by  the  method 
of  demidesiccation,  and  it  is  first  examined  in  salt  solution, 
being  afterwards  treated  with  dilute  acetic  acid  and  hsema- 
toxylin.  But  it  is  a  troublesome  matter  to  make  the  separation 
fine  enough  without  disturbing  too  much  the  arrangement  of 
the  cellular  elements  of  the  tissue.  Fortunately  we  can  obtain, 
from  the  tail  of  the  mouse  or  rat,  tendons  which  are,  so  to 
speak,  naturally  dissociated ;  for  fine  silk-like  tendons  run 
along  the  whole  length  of  the  tail,  and  can  readily  be  drawn 
out,  needing  no  further  manipulation  than  is  necessary  to  place 
them  advantageously  under  the  microscope.  The  following  is 
the  mode  of  procedure  : — 

In  a  recently  killed  mouse  the  tail  is  seized  about  half  an 
inch  from  the  tip  between  the  thumb-nail  and  fore-finger  of 
the  right  hand ;  and  the  delicate  skin  being  nipped  through 
and  the  vertebral  column  broken  at  this  point  by  the  pressure 
of  the  nail,  it  will  be  found  quite  easy,  the  base  of  the  tail 
being  fixed  by  the  left  fore-finger  and  thumb,  to  separate  the 
end  altogether  and  drag  it  away  from  the  remainder  of  the 
tail.  In  doing  so  it  will  be  found  that  the  minute  tendons 
which  are  attached  near  the  tip,  owing  to  their  comparative 
toughness  and  strength,  are  not  broken  through  at  the  spot  in 
question,  but  are  dragged  out  of  the  channels  in  which  they 
run,  and  may  in  this  way  be  obtained  in  the  form  of  a  bundle 
of  exquisitely  fine  silky  threads,  which  are  to  be  immediately 
immersed  in  a  glass  dish  of  salt  solution.  Now  cut  away  two 
or  three  of  the  fine  threads  with  sharp  clean  scissors,  and 
seizing  them  by  one  end  with  fine  forceps,  or  leading  them 
with  a  needle-point,  float  them  on  to  a  glass  slide  which  is 
held  immersed  in  the  fluid,  and  is  then  carefully  lifted  out. 
After  arranging  the  minute  tendons  as  nearly  straight  as 
possible  on  the  slide  and  blotting  up  most  of  the  superfluous 
salt  solution  or  allowing  it  to  run  ofl",  place  a  short  piece  of 
hair  parallel  with  them,  to  avert  the  pressure  of  the  cover-glass, 
which  is  now  placed  over  the  middle  of  the  threads  in  such 


TENDON  10!) 

a  way  that,  since  they  ;u'e  considerably  longer  than  tlie  width 
of  the  cover-glass,  their  ends  project  beyond  on  either  side. 
The  object  of  this  is  to  permit  them  to  l)e  drawn  straight 
should  the  superposition  of  the  cover-glass  have  displaced  them. 
These  ends,  moreover,  since  they  are  exposed  to  the  air,  soon 
dry  and  stick  to  the  slide,  so  that  subsequent  treatment  with 
reagents  does  not  tend  to  displace  the  tendons,  wluch  are  thus 
maintained  in  an  extended  condition.  Examined  thus  in  salt 
solution,  little  is  visible  beyond  the  slightly  wavy,  closely 
packed  white  fibrils,  collected,  as  longitudinal  streaks  seen  here 
and  there  indicate,  into  a  few  indistinct  bundles.  But  allow  a 
little  dilute  acetic  acid  (1  part  of  the  glacial  acid  to  200  of 
salt  solution)  slowly  to  pass  under  the  cover-glass,  and  a  re- 
markable change  becomes  apparent.  As  the  acid  reaches  the 
tendons  they  slowly  swell  up  and  become  more  transparent, 
the  fibrils  becoming  indistinct ;  and  now  chains  of  small  oblong 
faintly  granular  cells,  each  with  a  clear  nucleus  situated  near 
one  end  of  the  cell,  and  often  opposite  that  of  a  neighbouring 
cell,  come  into  view.  These  are  the  tendon-cells,  the  corpuscles 
of  the  fibrous  connective  tissue  ;  only  the  central  thicker 
portion  of  each,  which  lies  in  the  interstice  between  three  or 
more  tendon  bundles,  is  seen  at  present  ;  the  thin  lamellar  pro- 
longations, which  extend  between  two  tendon  bundles,  are  too 
delicate  to  be  made  out  without  staining.  In  some  of  the 
chains  a  bright  longitudinal  line  is  to  be  seen  on  each  cell  ; 
this  appeai'ance  is  merely  produced  by  a  lamellar  prolongation 
of  this  sort  which  happens  to  extend  vertically  to  the  plane 
under  observation. 

After  the  action  of  the  acid  has  been  prolonged  for  some 
time,  the  cells  gradually  lose  their  distinctness,  and  eventually 
can  with  difficulty  be  made  out,  although  the  fibres  are  more 
swollen  and  indistinct  than  ever.  But  if  a  little  carmalum  or 
haemalum  is  allowed  to  run  under  the  cover-glass,  first  the 
nuclei,  and  then  the  bodies  of  the  cells  and  their  prolongations 
become  coloured,  whilst  the  fibres  I'emain  unstained,  just  as 
in  the  parallel  case  of  the  areolar  tissue. 


110  PKACTICAL  HISTOLOGY 

When  the  colouration  is  sufficiently  deep,  the  staining 
fluid  may  be  replaced  by  water,  and  this  in  the  usual  way  by 
glycerine  and  water  ;  and  finally,  the  edges  of  the  cover-glass 
being  cemented,  the  preparation  can  be  permanently  pre- 
served. Examined  with  a  high  power,  the  tendon-cells  now 
appear  in  the  successive  horizontal  planes  as  quadrangular 
flattened  bodies,  thickest  near  the  middle,  and  gradually 
shading  ofl"  at  either  side,  and  marked  with  one  or  more  dark 
lines  ruiniing  longitudinally,  which  are,  in  fact,  the  bright 
lines  to  which  attention  was  previously  drawn,  and  which 
have  now  become  stained.  To  show  that  these  are  actual 
flattened  extensions  of  the  cell,  and  not  mere  markings,  it  is 
necessary  to  compare  the  appearances  presented  by  a  trans- 
verse section. 

Another  method  of  displaying  the  cells  of  tendon  is  that  origi- 
nally employed  by  Eanvier.  One  or  two  of  the  smaU  tail  tendons 
are  placed  on  a  slide,  and  their  ends  are  fixed  with  paraffin,  so  as 
to  keep  them  extended.  A  few  drops  of  a  1  per  cent,  solution  of 
picrocarminate  of  ammonia  are  then  placed  upon  them  and  left  for 
from  half  an  hour  to  several  hours,  after  which  the  picrocarmine  is 
washed  away  with  distilled  water  and  the  tendons  are  mounted  in 
glycerine,  acidulated  Avith  acetic  acid. 

Transverse  section  of  tendon. — To  obtain  this,  sections 
may  be  made  across  a  piece  of  the  tail  of  the  mouse  or  rat. 
For  observing  the  cells  the  tissue  should  be  stained  with  gold 
chloride  as  follows  : — A  short  piece  of  the  tail  deprived  of  its 
skin  is  immersed  for  five  minutes  in  formic  acid  solution 
(1  to  4).  It  is  then  placed  directly  in  gold  chloride  solutioil 
(1  per  cent.)  and  left  in  this  for  about  an  hour.  Then  it  is 
rinsed  with  water  and  replaced  in  1  to  4  formic  acid,  in  which 
it  is  left,  in  the  dark,  for  twenty-four  or  forty-eight  hours. 
It  is  now  thoroughly  washed  with  water,  soaked  in  weak 
gum,  frozen,  and  sections  are  cut  across  it  with  a  microtome. 
The  sections  should  be  mounted  in  glycerine. 

For  most  purposes,  however,  it  is  best  to  take  a  large 
tendon,  for  it  is  easier  to  get  transvei'se  sections  of  such  a  one, 


TKNDON  111 

and  in  all  essential  points  of  structure  it  is  quite  similar  to  the 
minute  tendons  which,  for  the  sake  of  convenience,  we  have 
just  been  employing.  A  piece,  tlien,  of  any  tendon,  large 
enough  to  be  grasped  by  the  fingers,  is  placed  in  strong  spirit 
for  a  day  or  two.  This  gives  it  a  very  hard,  horny  con- 
sistence, and  it  is  easy,  with  a  sharp  knife  or  razor  wetted  with 
spirit,  to  get  one  or  two  thin  sections  from  the  end.  These 
are  placed  in  aqueous  solution  of  magenta,  or  picrocarmine,  or 
in  carmalum  until  they  are  sufficiently  stained.  The  excess 
of  stain  is  then  washed  away  by  water  ;  and  finally  a  little 
diluted  glycerine  is  placed  upon  a  cover-glass,  which  is  inverted 
over  the  preparation. 

Another  and  perhaps  a  better  method  is  to  harden  the 
tendon  in  2  per  cent,  bichromate  of  potash  for  a  fortnight  or 
more,  and  after  soaking  in  gum  to  cut  sections  of  it  by  the 
freezing  microtome.  The  sections  may  be  stained  with 
carmalum  and  mounted  in  xylol  balsam  after  being  passed 
through  alcohol  and  clove-oil. 

It  will  be  seen  that  the  tendon  is  divided  into  fasciculi 
by  septa  of  areolar  tissue,  the  corpuscles  of  which  (seen 
edgeways)  are  brought  into  view,  being  stained  by  the  log- 
wood ;  it  will  further  be  observed,  if  the  sectional  area  of 
one  of  the  smaller  fasciculi  is  attentively  examined  with  a 
high  power,  that  it  again  is  divided  (although  incompletely) 
into  several  still  smaller  bundles  by  the  branching  processes 
of  deeply  coloured  stellate  bodies  situate  at  the  angle  of 
junction  between  three  or  more  such  bundles,  and  extending 
a  greater  or  less  distance  between  the  neighbouring  bundles. 
These  stellate  bodies,  with  their  processes,  are  the  tendon- 
cells  with  their  lamellar  extensions,  as  .seen  in  section ;  the 
smaller  fasciculi,  which  are  separated  by  areolar  tissue,  cor- 
respond with  the  whole  of  one  of  the  caudal  tendons  of  the 
mouse  or  rat. 

Cell-spaces  of  tendon. — To  show  the  cell-spaces  in  which 
the  above-mentioned  tendon -cells  are  contained  : — 

As  before,  break  off  the  end  and  draw  out  sc)n)e  of  the 


112  PRACTICAL  HISTOLOaY 

tendons  of  a  mouse's  tail  (that  previously  used  will  still 
yield  a  suE&cient  number).  Then  hold  the  tendons  in  a 
shallow  dish  of  distilled  water,  and  with  a  medium -sized 
camel-hair  pencil  brush  them  firmly  from  end  to  end  six  or 
eight  times.  Remove  them  now  from  the  water,  and  immerse 
them  in  a  large  watch-glass  of  nitrate  of  silver  solution 
(1  per  cent.)  for  five  minutes  ;  then  place  in  a  glass  vessel 
of  distilled  water  and  expose  to  sunlight.  As  soon  as  they 
are  brown,  pieces  may  be  cut  off,  laid  straight  in  glycerine 
upon  a  slide  and  covered  ;  after  which  the  cover-glass  may  be 
fixed  in  the  usual  way.  Or  they  may  be  placed  (stretched)  in 
alcohol  for  a  few  minutes,  then  passed  through  oil  of  cloves, 
and  finally  mounted  in  xylol  balsam. 

Epithelioid  covering  of  tendons. — The  object  of  first 
brushing  the  tendons  is  to  remove  the  layer  of  flattened  cells 
which  covers  the  surface  of  each,  and  which,  if  allowed  to 
remain,  prevents  the  silver  solution  from  properly  acting  upon 
the  deeper  parts  of  the  tissue.  To  show  this  layer,  another 
set  of  tendons  may  be  treated  with  the  silver  solution  in  a 
similar  way,  but  with  a  minute's  immersion  and  without 
previously  brushing  them,  when  it  will  probably  be  found 
that  the  superficial  epithelioid  stratum  is  alone  apparent. 

Adipose  tissue. — The  simplest  way  of  showing  the  fat- 
vesicles  is  by  teasing  out  a  small  portion  of  the  tissue  in  a 
drop  of  salt  solution  ;  taking  the  precaution  of  putting  a 
narrow  slip  of  blotting-paper  on  either  side  to  avert  the 
pressure  of  the  cover-glass. 

But  a  far  better  way  of  demonstrating  the  structure  of  the 
tissue  generally,  consists  in  the  employment  of  the  method  of 
interstitial  injection  with  gelatine  described  on  pp.  101,  102. 
The  gelatine  is  injected  into  the  interior  of  a  fat-lump,  and 
sections  are  made  and  treated  in  the  way  described  for 
areolar  tissue.  By  this  method  the  fat-cells  are  somewhat 
separated  from  one  another,  and  all  their  parts,  as  well  as 
the  intermediate  tissue  and  blood-vessels,  are  much  better 
displayed. 


ADIPOSE   TISSUE  113 

Fat  may  also  be  studied  by  the  eiuploynient  of  the  other 
methods  recommended  for  areolar  tissue,  especially  that  givea 
on  p.  98. 

Membrane  of  the  fat-cell. — To  show  this  distinctly 
Ranvier's  method  may  be  recommended.  An  interstitial 
injection  is  made  with  a  weak  solution  of  nitrate  of  silver 
(1  in  1,000),  and  a  minute  portion  of  the  fat-lump  thus 
rendered  cedematous  is  removed  with  scissors,  transferred  to 
a  slide  and  covered.  Many  of  the  fat-cells  exhibit  the  enve- 
lope and  nucleus  separated  by  a  distinct  space  from  the  fat- 
drop.  The  silver  solution  would  appear  to  have  penetrated 
by  endosmosis  and  to  have  become  collected  between  the  fat- 
drop  and  its  enclosing  membrane. 

Development  of  fat. — To  complete  the  study  of  adipose 
tissue,  the  fat-cells  should  be  observed  in  process  of  develop- 
ment. For  this  purpose  a  preparation  of  the  subcutaneous 
tissue  from  a  part  where  fat  is  being  deposited  may  be  obtained 
from  the  moderately  advanced  foetus  of  any  mammal.  The 
new-born  rat  is  especially  to  be  recommended,  since  in  its 
subcutaneous  tissue  there  are  generally  to  be  found,  not  only 
cells  which  are  in  every  stage  of  fat-deposition,  but  others  in 
addition  which  exhibit  the  formation  of  blood-vessels  and  the 
simultaneous  formation  within  the  same  cells  of  red  blood- 
corpuscles.  The  mode  of  preparation  is  very  simple,  all  that 
is  necessary  being  to  strip  the  skin  from  the  back,  snip  off 
with  scissors  a  little  of  the  gelatinous-looking  tissue  from  the 
borders  of  a  tract  where  the  fat  is  already  partly  deposited, 
and  to  place  the  portion  so  obtained  in  a  drop  of  salt  solution 
upon  a  slide,  and  cover  it. 


114  PEACTICAL    HISTOLOGY 


CHAPTER   IV 

CARTILAGE 

Articular  cartilage. — This  is  to  be  studied  in  sections 
made  both  parallel  and  vertical  to  the  surface. 

Study  of  living  cartilage. — From  an  animal  that  has  just 
been  killed  remove  one  of  the  limb-bones,  with  its  articular 
ends,  and  with  a  clean,  sharp  scalpel  or  razor  take  a  slice,  as 
thin  as  possible,  off  the  cartilage,  and  quickly,  before  it  has 
time  to  become  dry,  transfer  the  piece  to^a  drop  of  serum  upon 
a  slide,  place  a  cover-glass  over  the  preparation  and  examine 
with  a  high  power.  Turning  the  attention  more  particularly 
to  the  cartilage-cells,  the  arrangement  of  these  in  groups  in 
the  faintly  granulous  matrix  will  be  noticed.  Each  cell  is 
seen  to  be  provided  with  a  clear  round  nucleus,  which  in  some 
specimens  of  articular  cartilage  is  so  large  proportionately 
that  it  may  be  mistaken  by  an  inexperienced  observer  for  the 
whole  cell.  In  reality,  however,  the  cell-substance  is  repre- 
sented by  the  clear  material  (showing,  at  most,  a  few 
highly  refracting  granules)  which  lies  around  the  nucleus 
and  entirely  fills  the  cavity  or  space  in  the  cartilaginous 
matrix  in  which  the  cell  lies.  But  now  replace  the  serum  by 
distilled  water,  drawing  the  former  away  by  means  of  a  piece 
of  blotting-paper  placed  at  one  edge  of  the  cover-glass,  and 
allowing  a  drop  of  water  from  a  pipette  to  run  under  at  the 
opposite  edge  (see  fig.  12),  and  the  picture  soon  changes. 
Examine  the  cells  at  the  borders  of  the  slice,  for  these  are 
first  reached  by  the  water.  It  will  be  seen  that  the  clear  cell- 
substance  begins  to  be  separated  from  the  matrix,  and  acquires 


"x^: 


CARTILAGE  115 

a  jagged  outline,  fluid  collecting  in  the  interspace  which  is  now 
left.  This  clear  fluid  is,  there  can  be  little  doubt,  derived 
from  the  cartilage-cells  themselves,  and  not  from  the  water 
which  has  permeated  the  matrix.  The  most  probable  ex- 
planation of  its  appearance  is  that  the  cartilage-cells,  by 
virtue  of  a  certain  amount  of  vital  contractility  which  they 
retain,  shrink  on  contact  with  the  water,  as  they  do  on  the 
application  of  many  other  reagents  and  on  the  passage  of  an 
electric  shock,  and  that  the  clear  fluid  which  collects  around 
them  is  expressed  from  their  protoplasm  as  it  contracts. 
Whatever  the  explanation  may  be,  the  effect  is  this  :  that  the 
now  jagged,  shrunken  cell-body  assumes,  instead  of  the  clear 
aspect  which  in  the  fresh  condition  it  presented,  a  coarsely 
granular  appeai'ance,  so  much  so  indeed  that  the  nucleus 
wliieli  was  previously  so  apparent  is  now  entirely  obscured. 
Moreover,  as  already  indicated,  the  cartilage-cell  no  longer 
fills  the  cell-space  in  which  it  lies.  The  cells  always  undergo 
this  change  after  death,  unless  the  tissue  has  been  treated 
with  some  reagent  which  prevents  its  occurrence. 

In  preparing  a  specimen  of  cartilage  with  the  object  of 
permanently  preserving  it,  our  aim  should  be,  as,  indeed,  with 
every  tissue,  to  obtain  it  in  a  condition  and  form  as  nearly  as 
possible  approaching  that  which  it  had  whilst  living.  There 
are  numerous  reagents  which,  in  place  of  acting  like  water 
and  causing  contraction  of  the  cartilage-cells,  fix  them  in  the 
form  they  present  during  life.  Amongst  these  osmic  acid  may 
be  mentioned  first,  as  the  most  generally  valuable  reagent  which 
we  possess  for  this  purpose,  since  it  acts  in  like  manner  upon 
nearly  all  the  tissues.  Other  and  cheaper  reagents  serve  the 
purpose  equally  well,  however,  for  cartilage.  One  special  value 
it  certainly  has  ;  namely,  in  showing  that  the  little  granules  in 
the  protoplasm  of  the  cartilage-cell  are  many  of  them  of  a  fatty 
nature,  for  they  are  blackened  by  the  reagent.  A  1  per  cent, 
solution  of  alum,  and  a  saturated  solution  of  picric  acid  (both 
recommended  by  Ranvier),  preserve  the  cells  of  cartilage 
admirably.      But  the  best  and  most  convenient  reagent  for 


116  PEACTICAL  HISTOLOGY 

the  purpose  is  a  weak  solution  of  ehrouiio  acid  (1  part  to  500 
of  water). 

Structure  of  articular  cartilage. — The  articular  head  of 
one  of  the  long  bones  is  removed  from  the  recently  killed 
animal,  split  into  two  down  the  middle  with  a  strong  knife  or 
a  chisel,  and  the  halves  placed  in  a  large  quantity  of  a  solution 
of  chromic  acid  of  the  strength  above  indicated,  and  allowed 
to  remain  in  it  a  few  days.  The  exact  time  is  immaterial,  but 
in  specimens  which  are  left  rather  longer  in  the  liquid,  the 
bone  in  the  neighbourhood  of  the  cartilage  is  softened,  and  the 
cartilage- cells  are  moi-e  coloured  by  the  acid,  and  consequently 
more  apparent.  "When  it  is  desirtd  to  prepare  the  tissue  for 
the  microscope,  one  of  the  halves  is  taken  from  the  tluid, 
washed  in  water  for  a  minute  or  two,  and  then,  the  bone  being 
held  in  the  hand,  one  or  two  thin  sections  are  taken  from  the 
surface  of  cleavage,  and  therefore  vertically  to  the  articular 
surface,  including,  if  possible,  a  little  of  the  adjacent  bone. 
The  sections  are  placed  in  water  on  a  slide  and  covered,  and 
are  examined  first  with  a  low  power  to  study  the  general 
arrangement  of  the  cartilage  cell-groups  in  the  superficial, 
intermediate,  and  deep  strata  respectively,  and  subsequently 
with  a  high  powpr  to  see  the  intimate  structure  of  the  cells. 
These  should,  as  before  indicated,  present  as  nearly  as  possible 
the  same  appearance  as  during  life,  the  only  difference  being 
that  the  tissue  generally  is  less  transparent,  and  slightly 
coloured,  and  that  the  cell-outlines  are  rather  more  strongly 
marked.  These  ditlerences  become  less  obvious  when  glycerine 
has  been  permitted  to  difiuse  itself  under  the  cover-glass,  for 
the  preservation  of  the  specimen.  Previously,  however,  to 
the  addition  of  glycerine  the  preparations  may  be  stained  by 
luemalum  or  carn^ktm,  after  well  washing  them  with  water. 

Sections  are  next  to  be  taken  parallel  to  the  articular 
surface  and  mounted  in  the  same  manner  ;  but  it  must  be 
borne  in  mind  that  it  is  only  those  sections  which  include 
parts  of  the  cartilage  near  the  natural  or  artificial  surface 
which  will  be  of  value  as  respects  the  preservation  of  the 


CARTILAGE  117 

tissue  elements  in  their  natural  condition,  at  least  in  the  case 
of  the  thick  articular  cartilages  of  large  animals.  For  the 
preservative  solution  naturally  takes  some  time  to  permeate 
the  cartilaginous  matrix,  and  before  it  has  time  to  penetrate  to 
the  deeper  parts  the  cells  will  have  already  shrunk  away  from 
the  walls  of  the  enclosing  cavities  and  have  become  changed 
in  the  manner  previously  indicated.  So  that  the  deeper  sec- 
tions will  exhibit  merely  the  irregular,  contracted,  and  highly 
refracting  corpuscles  lying  loosely  in  their  cell-spaces. 

At  the  thinnest  parts  of  all  the  sections  cavities  may  be 
observed  in  the  matrix  which  are  devoid  of  cartilage- cells  ; 
these  having  dropped  out  in  the  process  of  preparation. 

Cell-spaces  of  cartilage.  Preparation  by  silver  nitrate. 
The  cavities  or  cell-spaces  of  cartilage  may  be  readily  shown 
by  the  same  method  as  was  employed  to  show  the  cell-spaces 
of  connective  tissue,  viz.,  treatment  with  nitrate  of  silver  and 
subsequent  exposure  to  the  light.  For  this  purpose  a  fresh 
joint  should  be  opened,  and  the  articular  end  of  one  of 
the  bones  (preferably  a  coni-ex  one)  removed  with  the  saw  or 
bone-forceps.  The  end  thus  removed  is  well  rinsed  in  dis- 
tilled water  and  then  transferred  to  nitrate  of  silver  solution 
(I  per  cent.),  in  which  it  is  allowed  to  remain  three  minutes. 
It  is  then  again  rinsed  in  water,  and  gently  brushed  with  a 
camel-hair  pencil  to  remove  adhering  silver  precipitate  ; 
after  which  it  is  placed  in  a  beaker  of  50  p.c.  spirit  and  exposed 
to  sunlight.  When  thoroughly  browned,  -ections  are  made 
from  the  surface  with  a  razor  wetted  with  spirit,  and  are 
placed  in  water  (care  being  taken  that  they  become  com- 
pletely immersed),  after  which  they  may  be  mounted  in 
glycerine  (or  in  xylol  balsam  after  having  been  passed  through 
alcohol  and  clove-oil).  ^ 

Some  of  the  sections  should  be  takM  W>m  near  the  edge 
of  the  cartilage  and  mounted  on  a  separate  slide.  These 
should  show  the  branched  cell-spaces,  which  present  a  trans- 
ition to  the  much  more  ramified  spaces  of  the  connective 
tissue  of  the  synovial  membrane. 


118  PEACTICAL  HISTOLOGY 

The  cells  themselves  which  occupy  the  spaces  are  not 
shown,  for,  as  in  silvered  tissues  generally,  the  cell-protoplasm 
remains  absolutely  unstained ;  indeed,  it  is  at  first  sight 
difficult'to  believe  that  the  rounded  cavities  which  are  seen 
really  contain  cartilage-cells.  But  their  nuclei  may  be  brought 
into  view  by  staining  with  haematoxylin,  subsequently  to 
the  matrix  being  coloured  by  the  nitrate  of  silver.  As  before 
mentioned  in  speaking  of  connective  tissue,  it  is  difficult, 
if  not  impossible,  at  the  same  time  to  bring  distinctly  into 
view  the  outlines  of  the  cell-spaces  by  the  silver  method,  and 
the  bodies  of  the  cells  themselves  by  some  other  method  of 

staining. 

Preparation  of  cartilage  with  gold  chloride. — The  cells 
of  cartilage  as  well  as  most  protoplasmic  structures  are  well  dis- 
played when  stained  by  the  gold  method.  This  is  applied  to 
cartilage  in  the  following  way.  Thin  sections  are  made  from 
the  articular  end  of  a  fresh  bone  and  are  placed  in  a  few 
drops  of  a  solution  of  chloride  of  gold  (1  part  in  200  of  water). 
After  half  an  hour  they  are  transferred  to  a  beaker  containing 
a  comparatively  large  amount  of  distilled  water  which  has 
previously  been  slightly  acidulated  with  acetic  acid,  so  as  just 
to  be  distinctly  acid  to  the  taste.  The  beaker  is  then  covered 
with  a  glass  plate  and  placed  in  a  window  in  a  warm  place, 
and  where  it  will  be  exposed  for  some  hours  a  day  to  sun- 
light. Here  the  sections  are  left  for  two  days,  after  which 
time  they  should  have  acquired  a  dark  violet  colour,  and  are 
ready  to  mount  in  glycerine.  But  before  this  is  done  they 
should  first  be  examined  with  a  low  power  in  a  drop  of  water  to 
see  whether  there  is  any  precipitated  matter  upon  the  surfaces 
of  the  sections.  If  this  is  the  case  it  must  be  brushed  away 
with  a  camel-hair  pencil,  the  sections  being  held  with  fine 
forceps  during  the  process.  When  finally  mounted  and 
examined  with  a  high  power,  the  cartilage- cells  should  retain 
precisely  their  natural  form  and  appearance,  except  that  they 
are  stained  of  a  violet  tinge,  whilst  the  matrix  remains 
almost  entirely  colourless.     In  sections  taken  from  the  edge 


CARTILAGE 


119 


of  the  cartilage,  the  ramified  transitional  cells  which  occupy 
the  corresponding  cell-spaces  shown  by  the  silver  method 
will  be  rendered  apparent. 

The  ensiform  cartilage  of  the  newt  is  thin  enough  to  be 
prepared  by  the  gold  method  without  the  necessity  of  cutting 

Fig.  50 


Warming  apparatus  for  maintaining  portions  of  tissue  exposed  to  the  light  at 
a  constant,  raised  temperature.  The  lower  part  of  the  apparatus  is  filled 
with  water,  into  which  the  gas  regulator  dips. 

into  sections.     But  it  is  well  to  strip  off  the  perichondrium 
before  the  preparation  is  mounted  in  glycerine. 

The  colouration  by  the  gold  and  silver  methods  appears  to  depend 
on  the  occurrence  of  a  deposition  of  the  metal  m  those  parts  of  the 
tissue  for  which  their  salts  have  the  greatest  affinity :  in  the  case  of 


120  PEACTICAL  HISTOLOGY 

gold  this  is  generally  the  protoplasm  of  the  cells  (and  the  nerve- 
fibres,  where  any  exist)  ;  in  the  case  of  silver  it  is  the  ground-sub- 
stance, or  matrix,  or  intercellular  substance  ;  so  that  the  results  of 
the  two  processes  may,  at  least  in  the  case  of  the  cartilage -cells,  be 
looked  upon  as  standing  to  each  other  in  the  light  of  a  positive  and 
negative  image,  using  the  terms  as  they  are  employed  in  photo- 
graphy. 

If  it  is  the  winter  time,  and  especially  if  there  is  very  little 
bright  sunlight,  it  is  of  importance  to  keep  the  beaker  of  acidulated 
water  in  which  the  tissue  is  placed  waron,  as  this  materially  facili- 
tates the  reduction  of  the  gold.  For  this  purpose  it  is  well  to  have 
some  arrangement  by  which  the  beaker,  or  several  of  them,  if  there 
be  much  of  the  same  sort  of  work  going  on,  can  be  kept  at  a  tem- 
perature of  about  30°  or  40°  Centigrade,  and  at  the  same  time 
freely  exposed  to  the  light.  Fig.  50  represents  a  convenient  appa- 
ratus for  this  purpose.  It  is  made  of  zinc  or  copper  with 
glass  sides  and  top,  and  may  be  fitted  with  a  gas  regulator  to 
prevent  the  temperature  from  rising  too  high.  When  used  for  the 
present  purpose  the  apparatus  should  of  course  stand  in  a  good 
light — in  a  window  if  possible. 

Costal  cartilage. — The  cartilages  of  the  ribs  and  those  of 
the  trachea  and  larynx  may  be  prepared  and  examined  in  a 
manner  similar  to  that  recommended  for  the  study  of  articular 
cartilage.  Sections,  both  parallel  and  vertical  to  the  surface, 
should  be  made  from  the  fresh  tissue  and  from  pieces  which 
have  been  in  chromic  acid  solution  ;  those  from  the  latter 
being  mounted  and  preserved  in  glycerine  as  before,  with  or 
without  previous  staining  with  hsematein. 

Structure  of  cartilage  matrix. — Sections  are  to  be  made 
of  a  piece  of  thyroid  cartilage  that  has  been  preserved  in 
spirit,  and  are  to  be  stained  with  logwood.  When  sufficiently 
coloured,  they  are  transferred  to  water  and  then  mounted  in 
glycerine.  The  logwood,  besides  staining  the  nuclei  of  the 
cells,  gives  the  matrix  also  a  deep  purple  colour.  But  this 
colouration  of  the  matrix  is  not  uniform,  for  some  parts 
become  stained  much  more  deeply  than  others  :  those  regions 
more  immediately  around  the  cells  and  cell-groups,  and  which 
therefore  are,  as  commonly  considered,  the  latest  formed  por- 


CARTILAGE  121 

tions,  having  apparently  more  affinity  for  the  colouring  matter 
of  logwood  than  the  otlier  and  oldei-  parts.  By  tliis  method 
the  whole  matiix  appears  marked  out  into  what  may  be 
termed  cell-territories,  although  series  of  definite  rings  can  l)y 
no  means  be  said  to  be  very  apparent  around  the  cell-groups. 

To  show  the  fibi'ous  structure  of  cartilage  matrix,  sections 
of  fresh  cartilage  are  macerated  for  some  days  or  weeks  in 
brine.  They  are  then  mounted,  and  pressure  is  made  upon 
the  cover-glass  so  as  somewhat  to  crush  the  sections.  A 
fibrous  appearance  may  thereby  be  rendered  apparent. 

Transition  to  yellow  fibro-cartilage. — If  the  arytenoid 
cartilage  of  the  ox  is  sliced  longitudinally,  it  exhibits  to  the 
naked  eye  in  its  lower  part  the  opaline  bluish  appearance  of 
hyaline  cartilage,  but  in  its  upper  part  the  faintly  yellow  aspect 
of  elastic  cartilage,  the  two  parts  being  separated  by  a  distinct 
line  of  demarcation.  Take  now  a  thin  section  from  the  cut 
surface,  including  a  little  of  both  parts,  and  mount,  if  fresh,  in 
alum  solution,  or,  if  the  cartilage  has  been  previously  hardened 
in  spirit,  at  once  in  diluted  glycerine,  coloui^ed  by  magenta.  It 
is  immediately  seen  that  the  cai^tilage  above  the  line  of  demar- 
cation merely  differs  from  that  below  by  the  superaddition  of 
a  network  of  comparatively  coarse  branching  (elastic)  fibres. 
Just  at  the  junction  of  the  two  parts  the  fibres  are  but  few  in 
number,  and  at  their  ends  often  imperfect — i.e.  they  may 
appear  continued  merely  as  rows  of  granules  as  if  not  yet 
fully  formed.  Further  into  the  yellowish  part  they  permeate 
the  matrix  very  thickly,  and  give  it  almost  a  granular  appear- 
ance, owing  to  many  of  them  being  seen  in  section.  But 
immediately  around  each  cell  there  is  usually  an  area  of  the 
matrix  entirely  free  from  fibres,  and  this  area  is  in  appearance 
exactly  like  the  matrix  of  hyaline  cartilage.  So  that  we  may 
conclude  that  yellow  or  elastic  cartilage  is  merely  to  be 
regarded  as  hyaline  cartilage  in  the  matrix  of  which  a  deposit 
of  elastic  substance  has  taken  place  in  the  form  of  branching 
fibres  ;  and,  indeed,  the  study  of  its  development  shows  this 
to  be  the  case. 


122  PKACTICAL  HISTOLOGY 

Yellow  fibro-cartilage. — A  tissue  may  now  be  studied 
which  is  composed  entirely  of  yellow  cartilage,  the  human 
epiglottis  or  the  external  ear  cartilage  of  the  ox,  for  example. 
A  section  of  epiglottis  stained  with  magenta  and  mounted  in 
dilute  glycerine  shows  what  at  first  looks  like  a  granular 
matrix  dotted  with  islands  of  hyaline  substance,  each  with  one 
or  two  cartilage-cells  in  the  centre.  The  granular  appearance 
of  the  matrix,  when  carefully  observed,  is  seen  to  be  due  to 
an  extremely  close  feltwork  of  fine  elastic  fibres  ;  in  fact,  the 
structure  is  quite  like  that  of  the  upper  part  of  the  ox's 
arytenoid,  although,  owing  to  their  fineness  and  closeness  in 
the  epiglottis,  the  individual  fibres  cannot  be  followed  for 
any  distance. 

White  fibro-cartilage  is  best  studied  in  sections  of 
intervertebral  disc  or  of  some  other  ligameat  which  is  composed 
of  this  tissue.  The  tissue  may  have  been  previously  hardened 
in  spirit.  Sections  should  be  made  both  parallel  with  and 
across  the  bundles  of  tendinous  fibres.  They  are  to  be 
stained  with  htemalum  and  mounted  in  glycerine.  It  will  be 
seen  in  the  longitudinal  sections  that  the  cartilage-cells  lie  in 
chains  between  the  tendon-bundles,  occupying  the  place  of  the 
ordinary  tendon-cells  ;  from  these  they  are  chiefly  characterised 
by  their  sharpness  of  outline  and  (in  transverse  sections)  the 
absence  of  lamellar  extensions,  as  well  as  their  greater  thick- 
ness ;  but  transitions  between  the  two  are  not  unfrequent, 
especially  near  the  tendon  insertion. 


123 


CHAPTER  V 

BONE 

Transverse  and  longitudinal  sections  of  hard  bone. — 

These  are  first  cut  as  thin  as  possible  with  a  fine  saw,  and  are 
afterwards  reduced  in  thickness  and  polished  by  grinding. 
The  bone  selected  should  be  thoroughly  macerated  and 
bleached  ;  it  should  be  absolutely  free  from  grease.  Sections 
may  be  made  from  different  bones,  flat  and  long,  but  for 
typical  specimens  of  the  compact  tissue,  transvei-se  and  longi- 
tudinal sections  of  one  of  the  long  bones  of  the  limbs — the 
ulna,  for  instance — may  be  recommended,  and  a  vertical 
section  of  one  of  the  flat  bones  of  the  skull,  such  as  the  parietal, 
should  also  be  prepared. 

The  first  thing  to  be  done  is  to  get  as  thin  a  piece  as 
possible  cut  with  a  fine  saw  from  any  desired  part.  The 
piece  so  obtained  is  ground  down  on  a  hone  wetted  with 
water.  The  hone  must  have  been  j^reviously  freed  from  all 
traces  of  greasy  matter  by  washing  with  soap  and  water  with 
a  little  soda  added.  The  piece  of  bone  is  pressed  down  and 
rubbed  to  and  fro  on  the  hone  simply  by  the  finger,  being 
ground  first  on  one  side  and  then  on  the  other.  The  feel  will  be 
almost  sufficient  to  tell  when  it  is  thin  enough,  and  this  may 
be  confirmed  by  placing  it  on  a  slide  without  covering  it  and 
examining  with  a  Ioav  power.  Numerous  scratches  will 
doubtless  be  visible  on  its  surface,  produced  by  the  grain  of 
the  stone,  however  fine  this  may  be  ;  but  unless  very  obvious, 
they  may  be  disregarded,  for  they  become  almost  invisible  in 
the  subsequent  preparation.    They  may,  if  desired,  be  removed 


124  PEACTICAL  HISTOLOGY 

by  polishing  the  section  upon  a  piece  of  plate  glass  with  the 
aid  of  putty  powder.  The  section  is  well  rinsed  in  clean  water 
by  aid  of  a  hair  pencil,  placed  upon  a  slide  to  dry,  and,  when 
dry,  mounted  in  Canada  balsam.  The  balsam  to  be  used 
for  this  purpose  must  not  be,  as  is  usually  the  case  with  that 
sold  in  the  shops,  semi-fluid,  but  quite  hard  in|j^the  cold — a 
condition  which  results  from  long  keeping,  or  may  be  pro- 
duced by  heating  a  little  of  the  more  recent  resin  in  a  capsule 
over  a  sand-  or  water-bath,  until  all  the  volatile  matters  are 
driven  off.  A  drop  of  the  melted  balsam  is  then  placed  upon 
a  slide,  which  is  warmed  over  a  flame  until  the  resin  has 
diffused  itself  pretty  evenly  over  the  central  part  of  the  slide. 
This  is  then  placed  on  the  table,  and  whilst  cooling,  but  before 
the  balsam  has  time  to  become  quite  hard  again,  the  thin  piece 
of  bone  is  placed  upon  it.  A  clean  cover-glass  is  then  taken 
up  by  the  forceps,  a  drop  of  the  balsam  placed  upon  it  also, 
warmed  in  like  manner,  and  quickly  inverted  over  the  prepara- 
tion. By  this  mode  of  proceeding  the  balsam  fills  up  and 
renders  invisible  the  scratches  on  the  surfaces  of  the  section 
•of  bone,  and  some  of  the  Haversian  canals  may  also  be  filled 
by  it ;  but  it  becomes  solidified  before  it  has  time  to  penetrate 
into  the  lacunae  and  canaliculi,  which  remain,  therefore,  filled 
with  air,  and  present  the  black  appearance  which  is  character- 
istic of  any  small  cavities  containing  air  when  they  are  viewed 
under  the  microscope  by  transmitted  light. 

Most  of  the  structural  points  with  regard  to  bone  can  be 
seen  much  better  in  these  preparations  of  the  hard  tissue  than 
in  sections  of  a  decalcified  bone.  But  it  is  important  in 
mounting  the  pieces  after  they  have  been  ground  down  to  be 
careful  that  the  balsam  does  not  remain  fluid  long  enough  to 
have  time  to  penetrate  into  the  thickness  of  the  section  ;  if 
this  should  happen,  the  whole  is  rendered  too  transparent  for 
many  of  the  details  of  structure  to  be  made  out.  On  the  whole 
it  is  perhaps  advisable  to  purchase  one  or  two  good  specimens 
rather  than  devote  a  large  amount  of  time  to  the  manufac- 
ture of  what  may  after  all  turn  out  to  be  but  an  indifferent 


BONE  125 

preparation.  Sections  of  hard  bone  and  of  teeth  are  amongst 
the  very  few  histological  preparations  which  are  usually 
better  made  by  those  who  make  a  business  of  preparing 
niit-roscopic  specimens  for  sale  than  Ijy  the  student  himself. 

Softened  bone. — In  addition  to  sections  of  hard  bone 
which  are  made  by  grinding  in  the  manner  above  described,  a 
portion  of  a  well-macerated  bone  may  be  placed  in  v.  Ebner's 
solution  (hydrochloric  acid  ii  grms.  ;  sodium  chloride  5  grms.  ; 
water  200  cc.  ;  alcohol  1000  cc.)  until  all  the  earthy  matter  is 
dissolved  ;  the  piece  is  then  steeped  in  70  p.c.  alcohol,  con- 
taining bicarbonate  of  soda,  to  free  it  from  the  remains  of 
the  acid,  and  finally  in  stronger  alcohol.  Sections  may  be 
made  with  a  microtome  and  mounted  in  glycerine  or,  after 
staining,  in  balsam. 

It  is  to  be  remembered  that  in  considering  the  structure 

of  a  bone  we  have  to  deal  not  merely  with  the  bony  matter 

pure  and  simple  and  its  included  cavities,  but  that  there  are 

in  addition  the  soft  contents  of  those  cavities — the  corpuscles 

in  the  lacunu>,  the  blood-vessels  in  the  Haversian  canals,  and 

the  marrow  in  the  medullary  cavity  and  in  the  interspaces  of 

the  spongy  tissue.     In  order  properly  to  view  these  structures 

(which  are  destroyed  in  a  macerated  bone)  we  must  emptoy  a 

reagent  which  will  at  the  same  time  decalcify  and  soften  the 

hard  matter  and  preserve  and  harden  the  included  soft  tissues.^ 

Such  a  reagent  is  to  be  found  in  a  solution  of  chromic  acid,  but 

as  the  decalcifying  powers  of  this  are  but  feeble,  the  portions 

of  bone  placed  in  it  must  be  as  small  as  possible,  and  a  large 

proportionate  amount  of  the  fluid  must  be  used.     For  example, 

if   the    bone    is  a  long  bone  about   the  size   of   the   human 

metacarpal,    a    piece   not  longer  than  a  quarter  of  an   inch 

should  be  sawn  from  it  and  placed  (suspended  by  a  thread) 

in    a   beaker  capable    of    containing    some    200    cc.    of    the 

chromic  acid  solution.     If  the    bone  is  larger,  it  is  well  to 

'  V.  Koch  has  devised  a  method  of  obtaining  by  grinding  thin  sections  of 
bones,  teeth  and  the  like  with  tlie  soft  parts  preserved  in  situ,  and  without  the 
necessity  for  decalcifj'ing.  The  method  is  largely  used  for  studying  tooth 
structure,  and  is  described  later  on  (p.  213). 


126  PEACTICAL  HISTOLOGY 

split  the  disc  thus  sawn  off  into  three  or  four  smaller  pieces, 
since  otherwise  the  decalcification  will  occupy  too  long  a  time. 
The  acid  used  should  be  at  first  very  weak  (1  in  500)  ;  in  two 
days'  time  this  may  be  changed  for  a  solution  of  1  in  200  ; 
and  this  again  in  another  two  days  for  1  in  1 00,  Beyond  this 
the  strength  of  the  acid  should  not  be  increased,  but  the  fluid 
should  be  renewed  every  three  days  at  least.  In  addition  to 
this  frequent  changing  of  the  liquid,  it  should,  throughout  the 
whole  time  of  softening,  be  stirred  as  often  as  possible  :  this 
is  of  importance,  for  every  agitation  brings  fresh  portions  of 
.acid  to  attack  the  earthy  matter  of  the  bone.  By  attention 
to  this  particular  the  time  which  the  pieces  take  to  become 
thoroughly  decalcified  may  be  materially  shortened.  The 
completion  of  the  process  is  ascertained  by  passing  a  needle 
through  the  middle  of  the  piece  employed  ;  if  it  meets  with  no 
gritty  obstruction,  all  the  earthy  matter  is  got  rid  of.  Should 
the  pieces  of  bone  be  thick  the  decalcification  process  may  be 
hastened  by  adding  to  the  chromic  acid  solution  a  little  nitric 
acid  (2  c.c.  to  each  100  c.c.  of  chromic  solution).  It  is  better 
not  to  employ  this  reagent  at  the  commencement  of  the  opera- 
tion, but  it  may  be  employed  during  the  later  stages,  especially 
in  conjunction  with  the  chromic  acid,  since  the  latter  reagent 
has  then,  by  its  coagulant  action  upon  the  tissue,  so  altered  it 
that  it  is  no  longer  swollen  by  the  dilute  mineral  acid. 

The  process  of  decalcification  being  completed,  the  pieces 
are  placed  in  running  water  for  a  day  or  two  to  get  rid  of  the 
excess  of  acid  imbibed,  and  are  then  transferred  to  spirit  fre- 
quently changed  ;  or  sections  may  be  made  at  once  without 
placing  the  piece  in  spirit  at  all.  The  sections  should  be  very 
thin  ;  they  are  stained  in  hpemalum,  carmalum,  or  magenta, 
washed  in  water,  and  finally  mounted  in  diluted  glycerine. 
The  corpuscles  within  the  lacunse,  at  least  their  nuclei,  are 
beautifully  shown,  and  all  the  soft  parts  are  more  or  less 
stain  ed,but  the  actual  substance  of  the  bone  is  only  slightly 
coloured.  At  the  same  time,  any  lines  of  demarcation  which 
may  be  present  indicating   successive    deposits   of    osseous 


BONE  127 

matter  in  the  formation  of  the  bone,  or  absorption  at  any  part 
and  subsequent  re-deposition,  with  the  characteristic  scolloped 
edge  which  such  a  junction  almost  always  possesses,  are  very 
clearly  shown  by  a  diffei-ence  in  the  colouration.  Moreover,  if 
in  the  section  there  happen  to  be  any  portions  remaining  of  the 
(ossified)  matiix  of  the  original  embryonic  cartilage,  this,  like 
cartilage  matrix  generally,  is  intensely  stained  by  logwood. 

Instead  of  chromic,  picric  acid  may  be  used  for  the  decal- 
cification. A  saturated  solution  of  the  acid  is  employed,  and 
care  is  to  be  taken  constantly  to  supply  fresh  crystals  of 
picric  acid  to  take  the  place  of  that  which  is  used  up  in  dis- 
solving the  lime  salts.  In  all  other  respects  the  proceeding 
is  much  the  same  as  for  the  chromic  acid  method,  except  that 
the  pieces  will  require  much  longer  washing.  This  is  to  be 
done  at  first  in  75  p.c.  alcohol  containing  lithium  car-bonate, 
and  afterwards  in  many  changes  of  alcohol.  The  sections  may 
be  stained  with  carmalum  or  with  picrocarmine  and  mounted 
in  glycerine  or  balsam. 

Two  other  methods  for  decalcifying  bone,  and  at  the  same  time 
preserving  the  soft  tissues,  may  be  mentioned.  The  first,  recom- 
mended by  Thoma,  consists  in  placing  the  bone  first  in  alcohol  for 
a  few  days,  then  in  alcohol  containing  1  part  in  6  of  hydrochloric  or 
nitric  acid,  and,  ^^•hen  softened,  agam  in  alcohol  containing  pre- 
cipitated calcium  carbonate,  for  two  weeks.  The  other  consists  in 
placing  the  tissue  direct  into  a  solution  made  by  dissolving,  with  the 
aid  of  heat,  1  gi-m.  of  pliloroglucin  in  10  c.e.  nitric  acid,  and  filling  up 
to  100  c.c.  with  water.  "When  decalcified  the  tissue  is  transferred 
to  alcohol.  The  decalcification  is  verj-  rapid  by  the  last  method  (a 
few  hoiurs),  and  the  tissues  stain  well  after  both  these  methods. 

Lamellae  and  Sharpey's  perforating-  fibres. — For  showing 
these  structures  macerated  bones  that  have  been  decalcified 
by  hydrochloric  or  nitric  acid  serve  very  well.  They  should 
before  use  soak  for  some  time  in  10  per  cent,  chloride  of 
sodium  solution,  which  is  occasionally  changed  to  get  rid  of  the 
last  traces  of  the  acid.  The  point  of  one  of  the  blades  of  a 
sharp  pair  of  forceps  is  then  inserted  obliquely  into  the 
bone  at  its  outer  surface,  and  a  small  piece  is  gripped  by  the 


128  PEACTICAL  HISTOLOaY 

other  blade  and  dragged  off  in  such  a  manner  that  it  pulls 
away  with  it  a  very  thin  strip  from  the  superficies  of  the  bone. 
A  few  such  strips  having  been  obtained,  either  from  different 
parts  of  the  bone  or  from  the  same  place  (at  different  depths, 
therefore),  they  are  placed  in  water  on  a  slide  with  the  inner 
surface  uppermost  and  examined  with  a  low  power.  In  some 
places  tapering  fibres  will  be  seen  projecting  from  the  surface 
of  the  torn- off  strip  like  nails  projecting  from  a  board  ;  in 
other  parts,  round  or  ovalish  holes,  corresponding  generally  in 
size  with  these  fibres,  will  be  apparent  :  these  are  apertures 
in  the  lamellse  where  the  latter  have  been  pierced  by  the  fibres 
of  Sharpey,  but  those  fibres  have  been  pulled  out  in  tearing 
off  the  strips.  Further,  there  may  be  made  out  a  faint  ap- 
pearance of  decussation  in  the  lamellae,  like  the  checks  on  a 
plaid,  but  oblique  in  direction,  best  marked  near  the  thin  edge 
of  the  strip  of  bone.  The  strips  may  be  mounted  in  dilute 
carbolic  acicl,  the  edges  of  the  coyer-glass  being  fixed  by  melted 
paraffin  and  then  by  one  or  two  layers  of  gold  size. 

The  bones  in  which  the  fibres  of  Sharpey  may  best  be  demon- 
strated in  the  way  above  described  are  the  flat  bones  of  the  skull. 
They  are  also  to  be  seen  both  in  shreds  and  in  sections  taken  from 
the  long  bones.  If  a  section  is  made  of  such  a  bone  at  the  place 
of  insertion  into  it  of  a  tendon  or  ligament,  and  in  the  direction  of 
the  fibres  of  the  tendon  or  ligament,  it  will  be  seen  that  the  bmidles 
of  fibres  of  the  last-named  structures  are  continued  into  the  sub- 
stance of  the  bone  as  perforating  fibres  or  fibres  of  Sharpey,  so  that 
these  in  fact  almost  compose  the  whole  of  the  osseous  tissue  at  this 
place  (Ranvier).  This  shows,  moreover,  that  the  fibres  of  Sharpey 
are  to  be  regarded  as  bundles  of  fibrous  tissue  (connected  either 
with  the  periosteum  or  with  a  tendon  or  ligament)  which  were 
intercalated  with  the  osseous  substance  proper  when  this  was 
formed  and  have  become  ossified  at  the  same  time.  When  tendons 
undergo  ossification,  the  bony  substance  which  is  formed  is  wholly 
of  the  same  nature  as  the  fibres  of  Sharpey ;  this  may  be  charac- 
teristically seen  in  the  ossified  tendons  which  are  met  with  in  the 
legs  of  birds.^ 

1  For  a  concise  account  of  methods  which  are  used  in  the  histological  in- 
vestigation of  bone  see  J.  Schaffer,  Zeitschr.f.  wiss.  Mikr.,  Bd.  x.  p.  167, 1893. 


I50NE   DEVELOPMENT  129 

Development  of  bone  in  membrane.— For  the  study  of 
the  intra-membranous  process  of  ossification  it  is  best  to  employ 
the  flat  bones  of  the  skull  of  sheep's  embryos  from  two  to 
three  inclies  long.  The  embryos  may  have  been  preserved  in 
bichromate  of  potash  (2  per  cent.),  or  spirit,  or  they  may  be 
employed  fresh.  A  piece  (corresponding  in  position  with  the 
future  parietal,  for  example)  of  the  still  membranous  skull- 
cap is  cut  out  with  tine  scissors,  placed  under  water  if  from 
bichromate  or  spirit,  under  salt  solution  if  recent,  and  the  skin 
and  muscular  layers  ai-e  torn  away  from  the  outside,  and  the 
dura  mater  and  cartilaginous  layer  (which  in  these  animals 
rises  up  laterally  from  the  cartilaginous  basis  cranii)  from  the 
inside.  The  membrane  in  which  the  bone  is  being  formed  is 
then  left.  It  is  held  upon  a  glass  slide  with  a  needle  or  fine 
forceps,  and  brushed  firmly  with  a  camel-hair  pencil  the  hairs 
of  which  have  been  cut  off  short  so  as  to  render  the  stump 
stiff  and  resisting.  The  piece  must  be  kept  wet,  and  examined 
from  time  to  time  with  a  low  power  to  see  whether  the  edo-es 
of  the  newly-formed  bone  are  sufficiently  clear  of  the  mem- 
brane and  corpuscles,  so  that  the  osseous  spicules  and  their 
fibrous  prolongations  are  readily  seen.  When  this  is  the  case 
the  piece  is  held  with  the  forceps  and  well  rinsed  in  water  or 
salt  solution  to  free  it  from  loose  particles  of  the  soft  tissue  ; 
it  is  then  placed  in  carmalum  solution,  and  when  sufficiently 
coloured  the  preparation  is  mounted  in  glycerine.  The 
osteoblasts  will  be  found  stained  by  the  dye  ;  the  ossified 
part  is  dark  and  highly  refracting,  the  osteogenic  fibres  by 
which  it  is  pi'olonged  remain  clear. 

The  process  of  intra-membranous  development  may  also 
be  very  advantageously  studied  in  sections  of  the  lower  jaw 
of  the  foetus  or  young  animal,  as  will  be  afterwards  pointed 
out  in  describing  the  mode  of  preparing  this  part  to  show  the 
development  of  other  structures,  such  as  the  teeth  and  hair 
(see  p.  216). 

Development  of  bone  in  cartilage. — To  study  the  later 
stages  of  ossification  in  cartilage  it  is  not   necessary  to  have 

K 


130  PKACTICAL   HISTOLOaY 

recourse  to  the  bones  of  a  foetal  animal,  since  any  which  are 
still  in  process  of  growth  will  serve  the  same  end,  and  it  is 
more  convenient  for  purposes  of  manipulation  if  they  have 
attained  a  certain  size.  The  long  bones  of  a  new-born  kitten 
may  advantageously  be  employed,  and  very  instructive  pre- 
parations may  be  obtained  from  the  recently  killed  animal, 
although  for  permanent  preparations  it  is  necessary  to  put 
them  into  a  fixative  and  hardening  fluid.  The  mode  of 
preparing  the  fresh  specimens  is  as  follows : — One  of  the 
long  bones — the  femur,  for  instance — is  removed,  and  its 
cartilaginous  head,  having  been  cleared  of  the  adherent  soft 
structures,  is  split  down  the  middle  with  a  strong  scalpel,  the 
split  extending  a  little  distance  into  the  subjacent  bone.  Then 
by  a  movement  of  the  scalpel  one  of  the  halves  is  broken  away, 
and  the  junction  of  the  bone  and  cartilage  is  brought  clearly 
into  view.  One  or  two  slices  as  thin  as  possible  are  now 
taken  from  the  surface  thus  produced,  including  the  line  of 
advancing  ossification,  and  are  placed  on  a  slide  in  salt  solution 
and  covered.  These  sections  are  of  course  parallel  with  the 
long  axis  of  the  bone  ;  but  to  complete  the  observation  others 
should  be  made  across  that  axis,  at  and  a  little  below  the  level 
of  the  line  of  ossification,  the  cartilaginous  head  being  first 
sliced  gradually  away  until  the  line  of  ossification  is  reached, 
and  then  a  series  of  sections  taken  of  the  part  of  the  cartilage 
which  is  undergoing  ossification  and  of  the  newly-formed  bone. 

Fresh  preparations  thus  made  are  both  very  beautiful  and 
instructive  if  the  sections  are  obtained  sufficiently  thin.  If  it 
is  wished  permanently  to  preserve  a  section  made  in  this  way, 
the  salt  solution  in  which  it  is  mounted  is  replaced  by  a  solu- 
tion of  osmic  acid  (1  per  cent.).  The  preparation  may  be  left 
in  this  for  an  hour,  after  which  time  the  osmic  acid  is  rinsed 
away  by  water,  and  finally  the  sections  are  mounted  in 
glycerine. 

Sections,  longitudinal  and  transverse,  of  ossifying  bones 
which  have  been  in  bichromate  of  potash  (2  per  cent.)  for  afew 
days  are  prepared  in  exactly  the  same  manner,  except  that  the 


MARROW  181 

treatment  with  osmic  acid  is  here  unnecessary.  The  prepara- 
tions can  be  mounted  at  once  in  glycerine. 

To  trace  all  the  steps  of  the  ossification,  it  is  requisite  to 
obtain  sections  of  bones  in  various  stages  of  formation.  For 
this  purpose  the  entire  limb  of  a  small  foetus  is  decalcified 
in  chromic  or  picric  or  other  acid,  in  the  same  way  as  has 
been  recommended  for  the  decalcification  of  pieces  of  the 
fully  developed  bone.  If  the  foetal  bones  are  still  small, 
the  time  necessary  for  such  decalcification  will  be  veiy 
much  shorter  than  was  requisite  for  the  dense  adult  bone,  nor 
need  the  strength  of  the  chromic  acid  solution  exceed  1  in  500. 
After  completing  the  hardening  process  with  spirit  the  limb  is 
stained  in  bulk  by  placing  it  overnight  in  0'5  per  cent,  solution 
of  magenta  in  spirit.  From  the  alcoholic  stain  it  is  transferred 
to  clove  oil  and  thence  to  xylol.  It  is  then  soaked  with  paraffin, 
and  sections  are  cut  parallel  to  the  longitudinal  axis  of  the 
limb,  the  sections  being  fixed  to  the  slide  and  mounted  in  xylol 
balsam  in  the  usual  way.  Other  sections  are  made  from  another 
limb  which  is  embedded  in  such  a  way  as  to  be  cut  across  the 
axis.  Nearly  all  stages  of  ossification  are  found  in  these  pre- 
parations, for  the  higher  limb  bones  are  usually  far  advanced  in 
process  of  formation  whilst  the  phalanges  are  only  commenc- 
ing ;  the  other  bones  being  generally  in  intermediate  stages. 
Magenta  produces  a  double  colouration  in  developing  bone, 
the  calcified  cartilage  being  stained  darkly,  the  periosteal  bone 
of  a  brighter  tint.  The  disadvantage  of  magenta  staining  is 
its  lack  of  permanence.  It  may  be  replaced  by  a  double  bulk 
staining,  first  with  solution  of  carmine  or  eosine,  and  after- 
wards with  hjematoxylin,  any  excess  of  the  latter  being 
removed  by  treatment  with  acid  alcohol  (see  p.  22).  The 
carmine-htematoxylin  stain  has  the  advantage  of  permanence. 

Medullary  tissue. — Ordinary  marrow,  which  consists  for 
the  most  part  of  adipose  tissue,  is  obtained  from  the  long 
bones  of  most  animals.  But  the  spongy  tissue  of  the  bones 
generally  and  the  medullary  canal  of  the  long  bones  in  some 
animals — the  rabbit  and  guinea-pig,  for  example — are  filled 

k2 


132  PEACTICAL  HISTOLOaY 

with  red  marrow,  which  contains  little  adipose  tissue,  but  is 
mainly  made  up  of  the  so-called  proper  marrow-cells,  which 
are  in  many  respects  similar  to  the  pale  corpuscles  of  the  blood 
and,  like  these,  exhibit  amoeboid  movements.  To  see  the 
marrow-cells,  therefore,  in  their  natural  condition  the  tissue 
should  be  taken  quite  fresh  and  examined  on  the  warm  stage. 
The  bone — the  femur  of  a  guinea-pig,  for  instance,  preferably 
a  young  animal — having  been  removed  and  cleared  of  the 
surrounding  soft  parts,  is  broken  across,  and  a  small  piece  of 
the  marrow  picked  out  and  broken  up  with  needles  in  a  drop 
of  serum  or  salt  solution  on  a  piece  of  thin  glass.  A  small 
piece  of  hair  is  then  added,  and  the  preparation  is  covered  :  a 
brush  dipped  in  oil  is  drawn  round  the  edge  of  the  cover-glass 
to  prevent  evaporation,  and  the  specimen  is  then  placed  on 
the  warm  stage  (p.  53)  and  examined.  The  different  sizes  and 
varying  forms  of  the  marrow-cells  are  to  be  noted,  as  also  the 
large  clear  nucleus  which  many  of  them  possess,  and  which  at 
once  distinguishes  these  cells  from  the  ordinary  white  blood- 
corpuscles.  But  many  are  also  observable  which  are  in  every 
respect  similar  to  the  latter,  and  may  indeed  not  improbably 
belong  to  the  blood,  for  the  blood-vessels  are  very  large  and 
numerous  in  the  medullary  tissue,  and  yield  the  numerous  red 
blood-corpuscles  which  are  seen  scattered  over  the  field. 
Besides  these  colourless  cells  of  the  marrow,  there  are  in  red 
marrow  a  number  of  similar  but  rather  smaller  elements  which 
resemble  the  marrow- cells  in  most  respects,  but  differ  from 
them  in  possessing  a  nucleus.  These  are  the  erythroblasts, 
which  are  characteristic  of  red  marrow.  They  are  in  fact 
nucleated  coloured  blood-corpuscles,  and  are  believed  to  give 
rise  to  the  ordinary  blood-discs  by  losing  their  nuclei  and 
becoming  modified  in  shape. 

Other  cells  may  occasionally  be  met  with  much  fewer  in 
number,  flattened  in  form,  and  sometimes  branched,  with  a 
large  clear  oval  nucleus,  and  in  some  instances  containing 
yellowish-red  pigment  granules.  They  are  larger  than  the 
proper  marrow-cells,  and  exhibit  no  changes  of  form.     There 


MARROW  133 

is  yet  another  element  to  be  found  in  the  marrow — most 
likely  to  be  met  with  in  a  bit  taken  from  near  the  inner 
surface  of  the  bone.  This  is  the  myeloplaxe,  and  is  charac- 
terised by  its  enormous  size — whence  the  name  yiant-cell — by 
its  granular  appearance,  and  by  containing  a  number  of  clear 
round  or  oval  nuclei  grouped  together  in  the  middle  of  the 
cell,  or,  in  many  cases,  a  single  large  irregular  nucleus  with 
numerous  buds  from  its  circumference. 

To  isolate  these  various  elements  of  the  marrow  better  than 
can  be  done  in  the  fresh  condition,  a  piece  of  the  tissue  is  to 
be  placed  in  one-third  spirit  for  a  day  or  two.  After  this 
time  a  small  portion  is  thoroughly  broken  up  with  needles  in 
a  drop  of  water  on  a  slide,  is  then  covered,  and  stained  by 
allowing  dilute  logwood  solution  to  flow  in  under  the  edge  of 
the  cover-glass.  The  logwood  is  replaced  in  a  minute  or  two 
by  water,  and  this  again  by  glycerine.  By  this  means  a  per- 
manent preparation  is  obtained,  which  can  be  studied  at 
leisure,  and  which  very  well  exhibits  the  different  kinds  of 
cells  above  enumerated,  whilst  the  red  blood-discs  are  rendered 
almost  completely  invisible.  For  class  purposes  the  method 
recommended  on  p.  81  may  be  adopted,  but  with  the  sub- 
stitution of  one-third  alcohol  for  Flemming's  fluid. 

Sections  of  marrow,  both  yellow  and  red,  should  also  be 
prepared.  They  are  made  either  from  alcohol  or  from  cor- 
rosive sublimate  preparations  (pp.  17  and  18),  and  the  pieces, 
after  hardening,  may  be  stained  in  bulk  with  carmalum, 
followed  by  alcohol  holding  picric  acid  in  solution,  or  by 
Heidenhain's  method  (p.  20).  For  the  study  of  the  develop- 
ment of  red  blood-corpuscles  from  erythroblasts  of  the  marrow 
it  is  best  to  make  thin  sections  from  corrosive  sublimate- 
hardened  marrow  of  birds,  and  to  stain  with  hsematoxylin 
and  eosin  or  carmalum  and  picric  acid. 

Cover-glass  preparations  of  red  marrow,  made  like  those  of  blood 
(p.  80),  except  that  the  marrow  is  smeared  over  the  cover-glass, 
also  give  valuable  results,  especiaUj'  for  the  study  of  the  granules 
of  the  marrow-cells. 


134  PEACTICAL  HISTOLOGY 


CHAPTER  VI 

MUSCULAR      TISSUE 

Involuntary  muscle. — It  is  easy  to  isolate  the  lanceolate- 
cells  of  which  this  tissue  consists.  For  this  purpose  all  that 
is  necessary  is  to  place  a  piece  of  any  organ  containing  plain 
muscular  tissue — the  intestine,  for  instance — in  a  weak 
solution  of  bichromate  of  potash  (one  part  to  800  of  water)  or 
in  one-third  alcohol  for  48  hours.  At  the  end  of  this  period 
of  maceration,  a  small  strip  of  either  the  longitudinal  or  the- 
circular  muscular  fibres  is  torn  off  with  forceps,  held  in  a  drop 
of  water  on  a  slide,  and  frayed  out  as  finely  as  possible  with  a 
needle.  A  cover-glass  is  then  laid  on,  and  the  preparation  is 
carefully  examined  with  a  high  power.  The  ends  and  edges 
of  the  larger  pieces  of  the  tissue  have  a  somewhat  ragged 
aspect,  due  to  the  projection  from  them  of  the  tapering  ends 
of  the  fibre- cells.  In  addition  to  these  partially  separated 
elements,  others  are  to  be  met  with  scattered  over  the  pre- 
paration which  are  wholly  free  and  in  which  all  the  charac- 
teristic appearances  of  this  tissue  can  be  distinctly  made  out. 
The  elongated  nucleus  in  the  middle  of  each  riband-shaped 
cell  can  be  seen  in  those  cells  which  lie  flat,  but  it  is  at  present 
indistinct.  It  may,  however,  be  brought  into  view,  as  can 
also  the  faint  longitudinal  striation  which  the  cells  exhibit,, 
by  employing  as  small  a  hole  as  possible  in  the  diaphragm  of  the 
microscope  to  admit  the  light  to  the  object.  But  to  show 
clearly  the  nuclei  of  the  plain  muscular  fibre-cells,  nothing  is 
l)etter  adapted  than  staining  the  tissue  with  a  weak  solution 
of   logwood.       It   must   be   used   quite  dilute,  and  suffered 


MUSCLE  135 

gradually  to  diffuse  itself  under  the  cover-glass  from  a  small 
drop  placed  at  one  edge.  It  is  not  a  good  plan  to  draw  it 
through  by  means  of  blotting-paper,  since  in  this  way  many  of 
the  isolated  elements  of  tlie  tissue  will  be  drawn  away  at  the 
same  time.  But  after  the  drop  of  logwood  solution  has  passed 
in  great  measure  or  entirely  under  the  cover-glass,  a  small 
drop  of  glycerine  may  be  added  at  the  same  spot  as  the  log- 
wood. This  as  it  diffuses  under  the  cover  will  gradually  push, 
as  it  were,  the  logwood  solution  before  it,  so  as  to  cause  the 
staining  fluid  to  traverse  successively  every  part  of  the  pre- 
paration, and  eventually  to  become  collected  entirely  at  the 
opposite  edge,  the  water  meanwhile  evaporating  and  leaving 
the  glycerine  in  possession  of  the  field.  All  that  is  needed  to 
complete  the  preparation  is,  in  the  course  of  a  day  or  two, 
to  fix  the  cover-glass  by  painting  a  little  gold  size  around  the 
edges.  Another  method  which  yields  good  results  is  to  place  a 
piece  of  the  tissue  in  picrocarmine  solution  and  leave  it  in  this 
for  some  days  or  even  weeks.  The  fibres  are  then  readily  iso- 
lated and  will  be  stained  yellow  by  the  picric  acid,  while  their 
nuclei  have  assumed  the  red  colour  of  the  carmine. 

The  involuntary  muscular  fibres  will  be  seen  in  section  in 
preparations  of  the  stomach  and  intestine  and  numerous  other 
organs,  so  that  it  is  not  necessary  to  make  special  pi'eparations 
at  this  stage  for  tlie  purpose  of  thus  showing  them.  It  may, 
however,  be  instructive  to  demonstrate  the  manner  in  which 
the  cells  are  applied  edge  to  edge  in  order  to  make  up  the 
bundles  and  lamellte  of  the  tissue.  This  is  often  shown  suffi- 
ciently well  in  a  thin  strip  which  has  been  stained  with  logwood 
and  mounted  in  glycerine  ^vithout  teasing. 

But  the  best  preparations  for  exhibiting  the  arrangement 
of  the  cells  are  those  stained  with  nitrate  of  silver.  As  in 
other  tissues,  this  reagent  stains  only  the  intercellular 
substance,  leaving  the  cells  themselves  uncoloured ;  their 
outlines  are  thus  brought  very  distinctly  into  view.  They 
may  be  demonstrated  in  the  frog's  bladder  by  the  following 
method  : — Cut  out  a  piece  of  the  bladder  and  lay  it  upon  a 


136  PEACTICAL  HISTOLOGY 

slide  with  the  inner  surface  uppermost.  Cover  it  with  water, 
and  with  the  finger  forcibly  rub  off  the  epithelial  lining,  wash- 
ing away  all  debris  with  a  stream  of  distilled  water.  Now 
cover  with  1  per  cent,  silver  nitrate  solution  and  leave  this 
on  for  two  minutes.  Wash  off  with  distilled  water,  and  place 
the  piece  of  bladder,  still  attached  to  the  slide,  in  a  dish  of  spirit 
and  expose  to  sunlight.  When  brown,  which  in  direct  sun- 
light will  be  in  a  few  minutes,  transfer  the  slide  to  clove-oil, 
and,  when  this  has  cleared  up  the  preparation,  dry  off  the 
excess  by  pressing  blotting-paper  upon  it  and  mount  in  xylol 
balsam.  Or  the  preparation  after  being  removed  from  the 
spirit  may  be  allowed  to  dry  on  the  slide,  and  when  completely 
dry  may  l^e  mounted  directly  in  xylol  balsam. 

Voluntary  muscular  tissue  :  cross-striped  muscle. — For 
the  examination  of  this  tissue  in  mammals  a  small  longitudinal 
shred  is  torn  or  snipped  off  from  any  muscle  of  the  limbs  or 
trunk  of  a  recently  killed  animal,  placed  upon  a  dry  slide,  and 
the  fibres  are  slowly  and  carefully  separated  from  one  another, 
one  by  one,  for  as  great  a  length  as  possible,  care  being  taken 
to  keep  them  moist  all  the  time  by  the  breath.  The  cover- 
glass  is  then  laid  on,  having  first  had  a  drop  of  serum  or  salt 
solution  placed  upon  it,  and  the  preparation  examined  first 
with  a  low  power,  to  study  the  shape  and  extent  of  the 
fibres,  afterwards  very  carefully  with  an  ordinary  high  power, 
and  eventually  with  as  high  a  power  as  it  is  possible  to  obtain. 
In  this  preparation  the  following  points  may  be  made  out  : 
(1)  the  delicate  sarcolemma  ;  (2)  the  muscle  nuclei  immediately 
beneath  it,  which  look  clear  and  oval  when  the  upper  surface 
of  a  fibre  is  exactly  focussed,  fusiform  when  seen  at  the  edge  ; 
(3)  the  dark  cross-stripes  of  the  muscular  substance,  seen  by 
careful  observation  with  a  very  high  power  to  be  pervaded  by 
fine  parallel  longitudinal  lines  ending  in  enlargements  within 
the  clear  stripes,  or  in  a  certain  focus  appearing  to  end  in  a 
dotted  line  in  the  middle  of  the  clear  stripes. 

The  structure  of  vertebrate  muscle  may  also  be  studied  in 
sections  of  alcohol-hardened  muscles,  the  sections  being  cut 


MUSCLE  137 

both  longitudinally  and  transversely,  stained  with  Imnnaluni 
or  picrocarniine,  and  mounted  in  xylol  balsam.  In  these 
sections  also  the  'muscle-spindles'  will  occasionally  be  met 
with  and  may  be  studied. 

The  sartorius  muscle  of  the  new-born  infant  furnishes  an 
especially  favourable  object  for  the  study  of  those  interesting 
structures  ;  but  they  are  to  be  found  in  most  muscles, 
although  in  some,  e.g.,  those  attached  to  the  globe  of  the  eye, 
they  have  not  been  detected.  They  appear  to  be  composed  of 
a  bundle  of  incompletely  developed  muscular  fibres,  enclosed 
in  a  sort  of  capsule  of  connective  tissue  containing  lymph- 
spaces,  and  having  medullated  nerve-fibres,  afferent  in  nature, 
ramifying  amongst  them  (Sherrington,  Journ.  PliysioL  xvii. 
237). 

Nuclei. — For  the  purpose  of  bringing  these  more  dis- 
tinctly into  view  another  piece  of  fresh  muscle  may  be  pre- 
pared as  just  described  and  mounted  in  dilute  acetic  acid 
(1  part  in  200  of  salt  solution).  It  will  be  found  that 
whereas  in  the  former  preparation  the  muscle-nuclei  could 
only  be  made  out  by  exercising  the  greatest  care  and  atten- 
tion, they  are  now  extremely  obvious,  studding  the  fibres  at 
intervals,  but  in  most  fibres  lying  at  the  surface  of  the 
muscle  under  the  sarcolemma.  If  a  frog's  muscles  are 
prepared  in  like  manner  with  acetic  acid  the  nuclei  are,  on 
the  contrary,  seen  for  the  most  part  to  be  embedded  in  the 
thickness  of  the  fibre,  and  this  is  also  the  case  with  some  of 
the  fibres  from  the  mammal  (red  muscles  of  the  rabbit). 

Sarcolemina. — This  is  extremely  delicate  in  mammalian 
muscle,  and  although  it  may  with  care  be  made  out,  is  never- 
theless much  more  easily  demonstrated  in  the  muscles  of  the 
lower  vertebrata — the  frog,  for  example.  With  this  end  a 
piece  of  fresh  and  living  muscle  is  carefully  separated  as 
above  described  and  covered  in  a  drop  of  salt  solution.  A 
process  of  disintegration  generally  begins  at  places  where 
the  fibres  have  been  touched  by  the  needles  in  the  process  of 
separation,  and  if   the  muscle  is  living   the  contractile  sub- 


138  PEACTIOAL  HISTOLOGY 

stance  breaks  and  shrinks  away  at  these  places,  leaving  clear 
sarcolemma  bridging  across  the  interval. 

Discs  and  muscle-columns. — In  order  to  exhibit  the 
manner  in  which  muscular  tissue  tends  to  break  up  into 
either  discs  or  columns,  according  to  the  nature  of  the  re- 
agent to  the  action  of  which  it  is  submitted,  two  pieces  of 
muscle  are  taken  from  an  animal  that  has  been  dead  some 
hours,  and  are  placed  for  a  day  or  two,  the  one  in  a  solution 
of  hydrochloric  acid  (1  in  500),  the  other  in  a  solution  of 
osmic  acid  (1  in  200).  A  small  portion  of  each  is  then  broken 
up  as  finely  as  possible  with  needles.  The  fibres  from  the 
hydrochloric  acid  are,  many  of  them,  found  to  cleave  into 
transverse  clear  discs,  some  of  which  will  be  noticed  lying 
flat,  others  seen  edgeways  ;  whereas  in  those  from  the  osmic 
acid  there  is  no  such  tendency  to  form  discs,  but,  on  the  con- 
trary, the  muscular  fibres  tend  to  break  up  into  longitudinal 
columns. 

Isolation  of  fibres. — In  order  the  better  to  compare  the 
fibres  either  of  the  same  or  of  difierent  muscles  as  regards 
length  and  diameter,  and  to  see  their  general  shape,  it  is 
necessary  to  isolate  a  number  of  them  in  their  whole  length. 
For  this  purpose  the  process  of  separation  by  the  aid  of 
needles  is  somewhat  tedious,  and  we  must  turn  to  reagents 
which  will  dissolve  the  intermediate  connective  tissue  which 
binds  the  fibres  together,  whilst  maintaining  them  intact. 
Such  a  reagent  is  to  be  found  in  a  solution  of  sulphurous  acid 
(liquor  acidi  sulphurosi  of  the  Pharmacopoeia).  The  muscle  is 
placed  for  a  week  or  more  in  a  well-stoppered  bottle  contain- 
ing a  considerable  quantity  of  the  acid,  and  is  kept  in  a  warm 
chamber  heated  to  about  40°  C.  This  facilitates  the  process  of 
maceration,  so  that  after  the  time  stated  a  mere  gentle  shak- 
ing of  the  bottle  is  sufiicient  to  cause  the  muscle  to  break  up 
in  great  measure  into  its  constituent  fibres.  Some  of  these 
may  then  be  removed,  placed  side  by  side  in  water  or  weak 
glycerine  on  a  slide,  and  covered,  with  the  usual  precautions 
to  obviate  the  pressure  of  the  cover-glass.     It  will  be  found 


MUSCLE  139 

tliat  the  muscular  substance  has  acquired,  in  consequence  of 
the  maceration,  a  granular  aspect,  and  that  the  usual  struc- 
tural appearances  are  for  the  most  part  indistinct.  But  the 
mode  of  preparation  may  be  nevertheless  employed  for 
purposes  of  measurement  and  comparison  of  size  and  form  of 
fibres  from  difterent  regions  of  the  body. 

Study  of  insect  muscle. — Many  insects  possess  two  kinds 
of  striped  muscular  tissue,  the  one  greyish  looking  and  com- 
posed of  large  tibres  having  the  same  appearance  under  the 
microscope  as  vertebrate  muscle,  except  that  the  characters 
are  more  distinct,  the  other  of  a  yellowish  aspect,  also  com- 
posed of  large  fibres,  which  have  a  less  marked  cross  striation 
than  the  others,  but  readily  break  up  into  longitudinal 
elements  (sarcostyles),  which  are  themselves,  however,  markedly 
striated.  Of  the  latter  kind  are  constituted  the  muscles  which 
move  the  wings  ;  of  the  former  the  muscles  of  the  trunk  and 
legs.  They  can  be  studied  both  in  the  living  condition  and 
after  preservation  in  alcohol. 

Examination  of  muscular  tissue  of  insects  in  the  living 
condition. — One  of  the  most  convenient  insects  to  employ  for 
the  purpose  is  the  common  gi'eat  water-beetle  [Dyticus  margi- 
nalis),  but  almost  any  beetle  or  other  insect  (especially 
the  wasp)  may  be  employed  instead.  The  head  is  first  cut  off 
and  the  trunk  is  then  split  longitudinally  with  scissors. 

"When  this  has  been  done  the  two  kinds  of  muscular  tissue 
will  be  noticed,  and  a  portion  of  each  can  be  taken  up  with 
the  point  of  a  scalpel,  rapidly  teased  on  a  slide  in  a  drop  of 
white  of  egg,  and  covered.  The  preparation  is  to  be  examined 
at  once  with  the  highest  available  power.  It  is  difficult  to 
make  out  the  details  of  the  structure  with  a  combination 
magnifying  less  than  1,000  diameters,  and  the  defining  power 
must  be  of  the  best. 

In  the  limb  muscles,  if  the  object  has  been  quickly  enough 
prepared,  numerous  fibres  will  be  found  which  show  the 
characteristic  successive  series  of  minute  longitudinal  lines  of 
interstitial  substance  running  through  the  darker  cross-stripes 


140  PEACTICAL   HISTOLOaY 

of  the  muscle,  and  prolonged  into  the  clear  stripes,  where 
they  pass  into  enlargements,  the  juxtaposition  of  which  in 
rows  side  by  side  gives  the  semblance  of  a  dotted  line  in  each 
clear  stripe,  single  or  double  according  as  the  j&bre  is  more  or 
less  extended.  Other  fibres  may  be  seen  in  which  the  dotted 
line  is  absent,  the  longitudinal  lines  not  being  enlarged 
within  the  clear  stripes  ;  and  these  show  especially  well 
the  muscle-columns  which  extend  in  the  direction  of  the 
length  of  the  fibre.  In  such  fibres  the  cross-striped  appear- 
ance is  indistinct,  due  to  the  fact  that  the  clear  strise  are 
but  slightly  marked,  their  bright  appearance  in  the  other 
fibres  being  greatly  enhanced  by  the  presence  of  the  rows 
of  strongly  refracting  dotlike  enlargements  of  interstitial 
substance. 

In  the  wing  muscles  many  of  the  muscle-columns  will  be 
isolated,  and  in  them  it  is  possible  to  make  out  the  trans- 
verse membranes  of  Krause  in  the  middle  of  each  clear 
stripe  and  in  the  more  extended  sarcostyles,  the  line  of  Hensen 
bisecting  each  dark  stripe. 

In  the  perfectly  fresh  preparations  of  the  insect's  muscular 
tissue,  spontaneous  waves  of  contraction  may  be  seen  passing 
from  end  to  end  of  many  of  the  muscular  fibres,  and  a  general 
idea  of  the  phenomena  which  accompany  the  contraction,  such 
as  thickening  of  the  part  of  the  fibre  at  the  time  the  wave  is 
passing,  and  approximation  of  the  cross-strise,  may  be  obtained. 
But  the  contraction  proceeds  too  quickly  for  all  the  details  of 
the  process  to  be  followed. 

Preparations  from  alcohol-hardened  muscle. — The  struc- 
ture of  musclecan  forthe  most  part  be  better  made  out  in  alcohol- 
hardened  preparations  than  in  those  fixed  by  any  other  method, 
and  the  alcohol  method  has  the  advantage  that  the  tissue 
can  be  stained  by  various  reagents  prior  to  being  mounted. 
One  only  of  the  modes  of  staining  need,  however,  here 
be  given,  viz.,  the  gold  method  of  Rollett,  which  is  a  special 
stain  for  this  tissue. 

It  M'ill  be  best,  before  proceeding   to    this,    to   examine 


ROLLETT'8   METIIOJ)  141 

teased  preparations  of  alcohol-haidened  insect  muscle  without 
staining.  To  fix  the  muscles,  the  insects  are  thrown  alive 
into  strong  spirit,  in  which  they  are  left  for  at  least  a 
day.  They  are  then  split  open  longitudinally,  and  portions  of 
the  two  kinds  of  muscle  removed  and  teased  (a)  in  dilute 
glycerine,  (b)  in  vinegar.  In  the  fornuir  the  sareostyles  are 
prominently  seen  ;  Imt  in  the  vinegar  preparation  the  sareo- 
styles become  swollen,  and  the  interstitial  substance  (sarco- 
plasm)  comes  distinctly  into  view  with  its  appearances  of  lines 
and  dots.  In  this  preparation  also  there  will  be  a  tendency 
for  the  fibres  to  break  across  into  discs,  and  these  discs,  when 
seen  on  the  flat,  exhibit  very  well  the  appearances  of  the  fibres 
as  seen  in  cross-section. 

RoUett's  gold  chloride  method  of  staining  muscular 
tissue.— A  small  piece  of  muscle,  from  either  the  ordinary  or 
the  wing-muscle  of  an  insect  which  has  been  in  90  per  cent, 
alcohol  for  from  twenty-four  to  forty-eight  hours,  is  steeped  for 
three  or  four  houi's  or  more  in  strong  glycerine.  From  this 
it  is  transferred  to  1  per  cent,  chloride  of  gold  solution,  and 
allowed  to  remain  for  from  fifteen  to  thirty  minutes.  It  is  then 
placed  in  formic  acid  (1  part  foi*mic  acid  to  3  parts  water),  and 
kept  in  the  dark  for  twenty-four  hours  or  more,  after  which  it 
may  be  teased  in  glycerine.  The  fibres  which  have  been 
prepared  by  this  method  show  many  varieties  of  staining, 
probably  dependent  upon  the  variable  time  they  have  been 
exposed  to  the  chloride  of  gold  (for  of  course  the  fibres  near 
the  surface  are  longer  exposed  to  the  reagent  than  those  nearer 
the  middle  of  the  piece  of  muscle),  and  some  will  be  found  the  in- 
terstitial substance  of  which  is  stained,  and  has  the  appearance 
of  a  fine  network  throughout  the  muscle,  others  in  which  this 
remains  colourless  while  the  sareostyles  are  stained.  In  the 
latter,  it  will  be  found  that  it  is  the  substance  of  the  dark  disc 
(sarcous  substance)  in  which  the  metal  is  deposited,  so  that  the 
sarcous elements  have  a  red  or  purple  colour,  while  the  substance 
of  the  clear  disc  is  left  completely  colourless.  In  these  pre- 
parations of  alcohol-hardened  muscle,  fibres  and  sareostyles  will 


142  PRACTICAL  HISTOLOGY 

be  found  in  varying  conditions  of  extension  and  contraction, 
so  that  the  phases  which  mark  the  passage  of  a  fibre  from 
the  state  of  full  extension  to  that  of  full  contraction  can  be 
observed. 

Examination  of  muscular  tissue  by  polarised  light. — The 
polarising  microscope  is  nothing  else  than  the  ordinary  micro- 
scope with  the  addition  of  two  Nichol's  prisms,  one  placed  below 
the  object  and  another  above  the  ocular  ;  the  upper  one  is 
generally  mounted  in  combination  with  a  low  ocular,  so  that 
it  is  not  necessary  to  use  the  ordinary  eye-piece.  The  light, 
coming  from  the  mirror,  becomes  polarised  as  it  passes  through 
the  lower  Nichol  (the  polariser).  If  now  the  upper  Nichol  (the 
analyser)  be  slowly  turned  round  as  it  is  being  looked  through, 
it  will  be  found  that  there  are  two  positions  in  which  the  field 
is  quite  dark  ;  that  is  to  say,  the  polarised  rays  are  entirely 
cut  off.  By  observing  now  the  relations  of  the  prisms  at 
these  positions  of  total  darkness,  it  will  be  found  that  their 
planes  of  polarisation — as  shown  by  the  way  in  which  the 
prisms  are  cut — are  at  right  angles  to  one  another.  In  all 
intermediate  positions  a  greater  or  less  amount  of  light  is 
enabled  to  traverse  the  analyser.  But  if  any  object  which 
possesses  the  property  of  refracting  light  doubly  is  placed 
upon  the  stage  of  the  microscope,  and  examined,  and  if 
then  the  field  is  made  dark  by  turning  the  analyser,  it  will 
be  found  that  the  doubly  refracting  substance  remains 
bright,  unless  it  happen  so  to  lie  that  its  optic  axis  is 
parallel  with  the  plane  of  polarisation  of  either  Nichol. 

The  observations  may  be  made  upon  the  living  or  upon 
alcohol-hardened  muscle.  The  portion  of  fibre  under  obser- 
vation should  be  quite  free  and  not  overlaid  by  other  fibres. 
The  change  in  the  optical  condition  of  the  fibres  which  ensues 
on  contraction  may,  if  due  care  and  patience  be  exercised,  be 
made  out.  The  results  arrived  at  by  the  examination  of 
portions  of  the  tissue  which  have  been  hardened  in  alcohol, 
and  mounted  in  glyceiine  or  Canada  balsam,  are  more  easily 
seen  than  if  living  muscle  be  examined. 


EXAMINATION   OF  MUSCLE   BY   POLARISED   LIGHT      143 

A  modification  may  be  made  by  sul^slituting  a  thin  piece 
of  mica  for  the  covering  glass.  This  causes  the  field  of  view 
to  become  tinted,  the  pai'ticular  colour  varying  with  the 
thickness  of  the  mica  and  the  relative  position  of  its  optic 
axis  to  those  of  the  Nichols,  and  any  doubly  refracting  sub- 
sbmce  which  is  now  examined  assumes  the  colour  which  is 
complementary  to  that  of  the  field.  The  object  of  the  re- 
volving stages  with  which  the  larger  microscopes  are  gene- 
rally fitted  is  to  enable  the  observer  to  modify  the  position 
of  the  optic  axis  of  the  tissue  which  is  being  examined 
with  relation  to  those  of  the  Nichols  ;  and  it  also  serves  when 
the  mica  is  used  to  change  in  like  manner  the  relative  position 
of  the  optic  axis  of  this  also,  and  thus  to  modify  the  colour  of 
the  field  of  view. 

Transversely  striated  muscle  is  not  the  only  tissue  which  is 
doubly  refracting,  for  the  property  is  possessed  by  the  Avhite 
fibrils  of  connective  tissue,  and  by  bone,  as  well  as  by  plain 
muscular  fibre-cells,  and  to  a  less  degree  by  ciliated  epithelium. 
But  it  is  the  only  one  which  exhibits  alternate  bands  of 
singly  and  of  doubly  refracting  substance.  It  has,  however, 
been  pointed  out  by  Ranvier  that  it  is  rather  the  conditions  of 
tension  of  a  tissue  than  differences  of  structui'e  which  tend  to 
determine  differences  in  the  optical  properties  of  the  substance 
of  which  it  may  be  composed.  Thus  he  instances  the  case 
of  cartilage,  the  matrix  of  which,  although  undoubtedly 
composed  of  the  same  substance  throughout,  becomes  doubly 
refracting  in  those  parts  where  the  cells,  either  from  pressure 
or  in  progress  of  growth,  assume  a  flattened  or  elongated 
shape,  singly  refracting  where  they  remain  rounded. 

Transverse  sections  of  insect  muscle. — Fairly  good 
views  of  the  sectional  appearances  of  the  substance  of  a 
muscle-fibre  may  be  accidentally  got  in  the  teased  preparations 
which  have  already  been  described.  But  to  obtain  complete 
sectional  views  of  the  muscles  pieces  of  hardened  muscle  are 
embedded  in  paraffin,  cut  very  thinly  with  a  microtome,  and 
mounted  by  the  adhesive  method.     Both  the  ordinary  and  the 


144  PEACTICAL  HISTOLOGY 

wing  muscles  of  insects  are  to  be  prepared  in  this  way  :  the 
pieces  of  muscle  may  be  stained  in  bulk  previous  to  embed- 
ding, or  on  the  slide  after  section. 

Termination  of  muscle  in  tendon. — If  the  tendons  of 
the  tail  of  a  mouse  or  rat  are  forcibly  drawn  out,  after 
nipping  off  the  end  of  the  tail  in  the  manner  described  in  the 
account  of  the  preparation  of  tendon  (p.  108),  it  will  generally 
be  found  that  portions  of  a  number  of  small  muscular  fibres  are 
adherent  to  each  tendon,  for  the  fibres  have  their  insertions 
into  the  tendon,  and  are  ruptured  by  the  force  employed. 
These  ends,  mounted  in  serum,  serve  conveniently  for  the 
study  of  the  mode  in  which  the  fibres  of  a  muscle  terminate  in 
a  tendon  when  the  fibres  of  the  latter  run  in  the  same  direc- 
tion as  those  of  the  muscle.  But,  easy  as  the  tissue  is  to  pre- 
pare, the  observation  is  complicated  by  the  fact  that  the 
muscular  fibres  form  generally  somewhat  of  a  clump  as  they 
pass  to  end  in  the  tendon.  The  preparation  may  be  improved 
by  being  stained  with  picrocarmine  solution.  This  colours 
muscular  tissue  yellow,  tendinous  tissue  red,  so  that  the  dis- 
tinction between  the  two  is  made  more  obvious.  It  is  best  to 
take  a  few  freshly  drawn-out  tendons,  and  to  mount  their  ends 
in  a  drop  of  the  picrocarmine  solution,  surrounding  the  edges 
of  the  cover-glass  with  melted  paraffin  to  prevent  evaporation 
of  the  liquid. 

Ranvier's  method.- — A  frog  is  pithed  and  is  placed  in  a 
litre  of  water  at  55°  C,  which  is  then  allowed  to  cool.  After 
a  quarter  of  an  hour  the  animal  is  removed  from  the  water, 
the  skin  detached  from  one  of  the  limbs  and  a  small  piece 
of  muscle  taken,  including  a  piece  of  its  tendon,  and  carefully 
dissociated  with  needles  in  a  drop  of  picrocarmine  solution  or 
in  serum  to  which  iodine  has  been  added.  It  will  be  found 
that  the  muscular  substance  has  for  the  most  part  retracted 
from  the  ends  of  the  sarcolemmal  sheaths,  and  that  in  favour- 
able cases  the  attachment  of  the  small  tendon  bundles  to  the 
ends  of  these  sheaths  can  be  made  out  without  much  diffi- 
culty. 


MUSCLE  145 

The  attachment  of  muscle  to  tendon  may  also  be  studied 
in  sections  of  a  muscle  passing  through  its  tendinous  attach- 
ment. 

The  blood-vessels  of  muscle  will  be  studied  later,  in  sections 
of  the  uijected  tissue,  after  the  methods  of  injecting  have  been 
described.  The  mode  of  termination  of  nerves  in  voluntary  muscle 
will  also  be  deferred  until  the  preparation  of  the  nerves  themselves 
has  been  treated  of. 


146  PEACTICAL  HISTOLOGY 


CHAPTER  VII 

NERVOUS   TISSUE 

MeduUated  nerve-fibres. — For  the  study  of  medullated 
nerve-fibres  a  piece  of  one  of  the  ordinary  nerves — those  of 
the  limbs,  for  example — may  be  cut  out  from  any  recently 
killed  animal.  If  the  nerve  is  a  large  one,  a  thin  strip  only 
should  be  used,  preferably  taken  from  the  interior  of  a 
funiculus  torn  longitudinally  into  two  pieces  by  fine  for- 
ceps. The  strip  is  to  be  placed  on  a  slide  in  a  little  serum  or 
salt  solution,  and  carefully  separated  as  finely  as  possible. 
This  separation  must  be  effected,  not  by  seizing  the  piece  any- 
where and  tearing  it  up  at  random,  but  by  inserting  fine 
needles  into  it  near  one  end  and  gently  drawing  them  asunder, 
so  that  the  piece  is  split  into  two.  Repeating  this  process  a 
number  of  times  on  the  resulting  pieces,  the  nerve  will  be 
eventually  separated  into  very  fine  bundles  of  fibres,  together 
with  a  number  of  more  or  less  isolated  fibres,  which  are  still 
nearly  straight  and  uninjured,  except  near  one  end.  The 
preparation  may  then  be  covered,  and  the  general  character 
and  appearance  of  the  fibres  investigated.  To  see  the  nodes 
of  Ranvier  well,  a  tolerably  large  fibre,  free  for  a  considerable 
part  of  its  length,  should  be  chosen,  and  by  moving  the  slide 
it  should  be  carefully  followed  with  an  ordinary  high  power. 
It  will  be  found  that  at  definite  and  not  very  close  intervals 
along  the  fibre  the  double -contoured  medullary  sheath  fails 
altogether,  and  the  axis-cylinder  alone  appears  to  continue 
the  nerve  at  these  points.  It  is  not  easy  to  see  the  oval 
nucleus  in  the  middle  of  each  segment  in  the  fresh,  unstained 


NERVE-1-MliKKS  147 

preparation,  but  the  oblifjuely  truncated  segments  into  whicli 
the  medullary  sheath  tends  to  split  are  very  obvious. 

Preparation  with  osmic  acid. — Osmic  acid  possesses  the 
property  of  staining  the  medullary  sheath  of  the  nerves,  which 
is  mainly  composed  of  lecithin,  of  an  inky-black  colour,  whilst 
the  other  parts  are  left  of  a  greyish  tinge.  Moreover,  the 
breaks  in  the  fatty  sheath — both  those  at  the  nodes  of  Ranvier 
and  the  irregular  breaks  between  the  medullary  segments — are 
by  this  means  brought  into  prominence,  owing  to  the  inter- 
mission of  the  darkly  stained  medullary  substance  at  those 
places.  At  the  same  time  the  axis-cylinder  can  be  distinctly 
made  out  crossing  the  intervals,  and  the  primitive  sheath,  or 
sheath  of  Schwann,  can  be  seen.  The  method  of  preparing 
these  osmic  prepai-ations  is  as  follows  : — From  an  animal  which 
has  been  quite  I'ecently  killed,  a  small  nerve,  or  piece  of  nerve- 
root,  not  larger  in  diameter  than  an  ordinary  thread,  is  chosen, 
and  a  piece  about  half  an  inch  long  is  cut  out,  but  in  doing 
so  care  must  be  taken  not  to  drag  upon  or  injure  the  nerve 
more  than  is  absolutely  necessary.  The  piece  is  placed  for 
four  hours  in  a  small  covered  glass  pot  containing  a  few  drops 
of  1  per  cent,  solution  of  osmic  acid  ;  it  is  then  transferred 
to  water  for  an  hour,  and  finally  placed  in  a  mixture  of 
glycerine  and  water  (equal  parts).  It  can  either  be  teased  in 
this  at  once  or  left  for  a  day  or  two  or  even  longer  ;  in  pre- 
paring it,  the  same  precautions  must  be  used  as  were  recom- 
mended for  the  preparation  of  the  fresh  nerve  (see  preceding 
paragraph). 

Fibres  of  E-emak. — To  see  the  grey  or  non-medullated 
fibres,  pieces  of  the  sympathetic  nerve  are  taken  fi-om  the 
neck  of  an  animal  and  prepared  and  examined  both  fresh  and 
after  ti'eatment  with  osmic  acid,  in  the  same  manner  as  the 
cerebro-spinal  nerves.  Many  small  medullated  fibres  are  here, 
however,  found  intermingled  with  the  non-medullated.  To 
obtain  non-medullated  nerves  almost  entirely  free  from 
medullated  it  is  best  to  make  preparations  from  the  splenic 
nerves,  which  can  be  readily  got  at,  and  in  some  animals  are 

l2 


148  PEACTICAL  HISTOLOGY 

comparatively  large.  Non-medullated  fibres  should  be  fixed 
as  rapidly  as  possible  after  death,  as  they  quickly  undergo 
structural  changes. 

Mode  of  union  of  the  nerve-fibres  to  form  the  nervous 
cords. — In  the  several  teased  preparations  both  of  the 
cerebro-spinal  and  of  the  sympathetic  nerves  there  will  be  seen, 
especially  in  those  prepared  with  osmic  acid,  besides  the 
actual  nerve-fibres,  in  the  first  place,  a  quantity  of  connective 
tissue,  for  the  most  part  of  the  nature  of  areolar  tissue,  which 
formed  a  general  ensheathment  for  the  nerve,  and  sends  par- 
titions in  between  its  several  bundles  or  funiculi  ;  secondly, 
the  special  sheaths  of  the  funiculi,  which  become  torn  and 
stripped  away  in  the  process  of  teasing,  and  which  look  like- 
flat  bands  composed  of  an  almost  homogeneous  substance,  but 
are  pervaded  by  a  network  of  fine  fibres  and  with  round  or 
oval  nuclei  scattered  upon  them  here  and  there.  Thirdly, 
there  will  be  seen  running  along  close  to  and  surrounding  the 
nerve-fibres  themselves  very  delicate,  nearly  straight  fibrils 
of  connective  tissue,  with  here  and  there  the  nucleus  of  a 
connective  tissue  corpuscle.  These  three  forms  of  connective 
tissue  represent  respectively  the  epineurium,  or  outer  sheath  ; 
the  perineurium,  or  funicular  sheath  ;  and  the  endoneurium, 
or  tissue  within  the  funiculus.  Their  relative  situation  and 
arrangement,  as  well  as  the  lamellated  structure  of  the  peri- 
neurium, can  only  be  properly  displayed  by  transverse  sections 
of  a  nerve-trunk. 

But  the  cell-outlines  on  the  lamellse  of  the  perineurium 
may  be  shown  by  the  silver  method.  For  this  purpose 
either  a  very  small  nerve  is  chosen,  and  a  piece  of  it  is  removed 
and  immersed  for  five  minutes  in  half  per  cent,  silver  nitrate 
solution,  after  which  it  is  washed  in  distilled  water  and  exposed 
in  glycerine  to  sunlight,  or,  should  it  be  wished  to  prepare 
a  larger  nerve  consisting  of  more  than  one  funiculus,  this  is 
partially  dissociated  in  a  few  drops  of  the  silver  solution, 
and  after  a  like  treatment  is  also  mounted  in  glycerine  and 
exposed  to  the  light.     After  a  few  minutes'  exposure  to  sun- 


NERVE  149 

light  the  preparations  may  be  examined.  It  will  be  found 
that  tlie  sheath  of  each  funiculus  or  nervous  bundle  is  covered 
by  large  epithelioid  markings,  and  if  the  preparation  is  suc- 
cessful, two,  three,  or  even  more  layers  deep  of  such  mark- 
ings may  be  counted  by  examining  with  a  high  power  and 
carefully  adjusting  the  microscope. 

In  addition  to  this  it  will  generally  be  found  that  any 
medullated  nerve-fibres  to  which  the  silver  solution  has  pene- 
trated are  marked  transversely  at  each  of  the  nodes  of  Ranvier 
by  a  dark  line,  or  rather  a  perforated  disc,  surrounding  the 
axis-cylinder.  These  markings  appear  to  be  owing  to  the 
•existence  of  a  substance  here,  between  the  segments  of  the 
nerves,  Avhich,  like  the  intercellular  substance  elsewhere,  has 
an  affinity  for  the  metal.  Finally,  at  many  of  the  nodes  of 
Ranvier,  particularly  if  the  immersion  in  the  silver  solution 
had  been  rather  prolonged,  the  latter  will  have  penetrated  as 
far  as,  and  will  have  become  reduced  in  the  substance  of  the 
axis- cylinder,  which  therefore  will  here  have  the  appearance  of 
a  dark  cylindrical  rod  piercing  the  ring  of  intersegmental  sub- 
stance, the  two  together  having  the  semblance,  under  a  mode- 
rate power,  of  a  little  black  cross  upon  the  nerve  at  those  points. 
In  such  preparations  the  axis-cylinder,  where  stained  at  the 
nodes  by  the  I'educed  silver,  has  a  cross-striated  appearance. 

Sections  of  a  nerve-trunk. — To  prepare  a  nerve-trunk  for 
microscopic  section,  it  should  be  hardened  in  picric  acid,  osmic 
acid,  Flemming's  fluid,  or  bichromate  of  potash.  It  should  be 
gently  extended  upon  a  piece  of  cork,  its  ends  being  fixed 
with  glass  pins,  and  it  is  best,  if  the  nerve  is  large  enough,  to 
ensure  rapidity  of  hardening  by  introducing  some  of  the  har- 
dening fluid  into  the  interior  of  the  nerve-trunk  by  a  glass 
pipette  with  a  capillary  point.  The  piece  is  left  in  picric 
acid  (saturated  solution)  for  two  days,  and  then  ti'ansferred 
to  spirit ;  in  1  per  cent,  osmic  acid  for  24  hours,  then  washed 
with  water  for  two  hours  and  transferred  to  spirit ;  in 
Flemming's  solution  for  two  days,  and  then  transferred  to 
spirit  •  or  in  bichromate  of  potash  for  seven  days,  and  then 


150  PEACTICAL  HISTOLOGY 

transferred  for  three  days  to  a  mixture  of  2  per  cent, 
bichromate  potash  (2  parts)  and  1  per  cent,  osmic  acid 
(1  part).  This  piece  also  is  placed  in  spirit  prior  to  embedding. 
Sections  may  then  be  cut  either  by  the  freezing  or  paraffin 
method,  and  mounted  either  in  glycerine  or  in  xylol  balsam, 
but  the  sections,  except  tliose  from  the  nerve  hardened  in 
osmic  acid,  must  first  be  stained  with  aniline  blue-black,, 
with  hsematein,  or  with  carmalum. 

Study  of  degenerating  nerve-fibres. — The  changes  which 
medullated  nerve-fibres  undergo  in  consequence  of  severance 
from  their  cells  of  origin  (which  in  the  case  of  the  sensory 
fibres  of  the  spinal  nerves  are  situated  in  the  ganglia  of  the 
posterior  roots,  and  in  the  case  of  the  motor  fibres  of  the 
spinal  nerves  are  placed  in  the  grey  matter  of  the  spinal  cord) 
may  be  studied  in  the  peripheral  part  of  nerves  which  have 
been  cut  from  two  days  to  three  weeks  previously,  the  nerves 
being  treated  in  pi'ecisely  the  same  way,  both  for  teased  pre- 
parations and  for  sections,  as  normal  nerves. 

Nerve-  and  ganglion-eells  ;  neuroglia-cells. — To  study 
the  exact  form  and  appearance  of  the  tissue  elements  of  the 
nervous  centres  they  may  be  isolated  and  examined  in  teased 
preparations,  although  their  position  and  local  relations  are 
best  made  out  in  sections  of  the  several  organs  in  which  they 
occur.  For  the  present  we  will  confine  ourselves  to  su 
description  of  the  best  methods  for  isolating  the  cells.  For 
this  purpose  the  tissue  is  treated  similarly,  from  whatever  part 
of  the  brain  or  spinal  cord  the  piece  to  be  examined  is  removed ;. 
but,  as  a  typical  example,  a  piece  of  the  spinal  cord  from  the 
lumbar  region  may  be  taken,  for  the  nerve-cells  are  here  very 
numerous,  and  consequently  more  readily  found  than  in  the 
other  regions. 

Cells  of  nerve-centres. — The  human  spinal  cord  is,  if 
obtainable,  best  adapted  for  the  study  of  the  cells,  for  they 
are  readily  picked  out  under  the  dissecting  microscope,  in  con- 
sequence of  the  little  mass  of  dark  pigment  which  each  con- 
tains.    If  a  piece  of  the  human  spinal  cord  cannot  be  got,  the 


NERVE-CELLS  151 

spinal  uianow  of  the  ox  or  calf,  or  some  other  large  animal, 
may  be  employed.  Small  pieces  of  tissue  are  dug  out  fi-on; 
the  grey  matter,  preferably  from  that  of  the  anterior  cornu, 
and  placed  in  one-third  alcohol.  After  two  or  three  days' 
maceration  in  this,  the  pieces  are  shaken  up  in  a  test-tube 
with  some  of  the  iluid  and  allowed  to  subside.  The  super- 
natant fluid  is  then  decanted  off,  fresh  one-third  alcohol  added, 
and  the  shaking  and  decantation  repeated.  Some  of  the 
debris  may  now  he  transferi'ed  to  a  slide,  a  piece  of  hair  placed 
in  the  fluid,  and  the  co\er-glass  gently  superposed.  The  cells 
may  then  be  sought  with  a  low  power  and  carefully  examined 
Avith  a  higher  one. 

If  the  preparation  is  successful,  and  it  be  desired  to  pre- 
serve it,  keeping  the  cells  at  the  same  time  as  much  as  possible 
of  their  natural  aspect,  the  best  plan  to  adopt  is  to  allow  a 
drop  or  two  of  a  1  per  cent,  solution  of  osmic  acid  to  flow  in 
at  the  edge  of  the  cover-glass,  and  after  it  has  acted  upon  the 
tissue  for  an  hour,  by  which  time  the  cells  will  have  become 
stained  of  a  dark  greyish  tint,  to  carefully  run  through  from 
the  same  side  first  a  little  distilled  water  to  wash  away  what 
remains  of  the  acid,  and  then  glycerine  to  preserve  the  pre- 
paration, after  which  the  edges  of  the  cover-glass  may  be 
cemented.  Or  the  cells  may  be  stained  with  picrocarmine  in 
the  test-tube  before  being  mounted.  For  this  purpose  the 
supernatant  one-third  alcohol  is  poured  off,  and  picrocarmine 
solution  added  and  kept  in  contact  with  the  debris  for  several 
hours  or  days.  Besides  neuroglia- cells  and  nerve-cells,  which 
in  a  carefully  prepared  specimen  may  be  very  well  seen,  with 
their  long  branching  processes  extending  in  some  cases  far 
beyond  the  field  of  the  high  power  objective,  some  points 
in  the  structure  of  the  nerve-fibres  of  the  spinal  cord  can  be 
well  made  out  in  these  prepai'ations.  Thus  fibres  may  be  readily 
found  in  which  the  medullary  sheath  has  broken  away  in  many 
parts  from  the  axis-cylinders,  and  where  not  actually  re- 
moved has  become  swollen  and  coagulated  in  irregular  masses 
around  the  axis-cylinder ;  changes  which  could  hardly  have 


152  PEACTICAL  HISTOLOGY 

taken  place  were  there  any  such  structure  surrounding  the 
nerve-fibres  as  the  primitive  sheath  of  the  peripheral  nerves. 
Further,  where  the  axis-cylinders  are  in  this  manner  laid  bare, 
as  they  often  are  for  a  considerable  part  of  their  length,  their 
fibrillar  structure  can,  with  a  high  power,  be  made  out  without 
difl&culty.  A  similar  structure  can  also  be  seen  in  the  pro- 
cesses of  the  nerve-cells,  and  extending  from  them  through 
the  body  of  the  cell  itself. 

Cells  of  ganglia. — Cells  from  ganglia,  whether  spinal 
or  sympathetic,  are  isolated  in  a  manner  similar  to  that 
employed  in  the  case  of  the  spinal  cord,  except  that  the  period 
of  maceration  in  the  one-third  alcohol  may  be  longer,  owing 
to  the  much  larger  amount  of  connective  tissue  by  which  the 
nervous  elements  are  invested.  Prolonged  maceration  in 
osmic  acid  (1  to  500  of  water)  is  also  a  method  of  much  value 
for  ganglion-cells.  It  is  possible  to  get  a  certain  number 
of  the  cells  sufficiently  isolated,  even  from  a  fresh  ganglion, 
without  any  maceration  ;  for  each  cell  being  loosely  contained 
in  a  special  capsule  of  flattened  cells,  it  readily  falls  out  when 
the  nerve  fibres  with  which  it  is  connected  are  ruptured. 
But  although  the  cells  themselves  of  the  ganglia  are  readily 
enough  separated,  it  is  more  difficult  both  in  the  fresh  and  in 
the  macerated  preparations,  to  show  their  continuation  to  the 
nerve  fibres,  and  the  T-shaped  branching  of  these.  The  mode 
of  permanently  preserving  the  specimen  is  like  that  employed 
in  the  preceding  preparation. 

Study  of  nerve-cells  by  the  silver-chromate  method. — 
Nerve-cells,  whether  in  nerve-centres  or  in  the  ganglia,  may  be 
displayed  with  the  cell-body  and  all  its  processes  of  an  intense 
black  colour  by  the  method  of  Golgi,  modified  by  Cajal  : — A 
small  piece  of  the  organ  to  be  investigated,  not  larger  than 
half  a  pea,  and  preferably  taken  from  a  developing  animal,  ^  is 
placed  fresh  in  a  considerable  quantity  (not  less  than  20  c.c.)  of 

^  The  spinal  cords  of  older  chick  embryos  and  of  new-bom  mice  or  rats 
are  very  well  adapted  for  the  purpose,  and  can  be  prepared  within  a  piece  of 
the  vertebral  column. 


METHOD   OF  GOLGI  153 

a  bichromate  osmic  solution  freshly  made  by  )nixing  o  volumes 
of  3  per  cent,  bichromate  of  potash  solution  with  1  volume  of 
1  per  cent,  osmic  acid.  The  piece  is  left  in  this  mixture  from 
1  to  5  days/  preferably  at  a  temperature  of  25°  C.  and  in  the 
dark,  and  is  then  dried  with  blotting-paper  and  transferred  to  a 
little  0-75  per  cent,  solution  of  silver  nitrate.  After  being  in 
this  for  a  few  minutes,  the  piece  is  placed  in  a  larger  quantity 
of  the  silver  solution,  to  which  1  drop  of  formic  acid  is  added  to 
every  100  c.c.  or  200  c.c.  In  this  the  piece  is  left  for  from  24 
hours  to  6  days.  It  is  then  placed  in  96  per  cent,  alcohol  for 
half  an  hour,  after  which  it  may  be  either  placed  in  collodion 
for  a  few  minutes,  and  by  this  fixed  upon  a  brass  holder  and 
cut  into  sections  with  a  microtome,  or  it  may,  if  fairly  hard 
and  if  large  enough,  be  simply  held  in  the  fingers  and  sections 
made  with  a  good  razor  wetted  with  96  per  cent,  alcohol. 
The  sections  are  passed  quickly  through  clove-oil  and  xylol,  and 
mounted  without  a  cover-glass  in  thick  xylol  balsam  or  xylol- 
dammar.  As  a  general  rule  they  need  not  be  cut  thin,  for  the 
greater  part  of  the  tissue  remains  unstained  and  only  a  few  of 
the  nerve  and  neuroglia  cells  are  coloured.  It  is  on  this  account 
that  it  is  easy  to  follow  the  processes.  It  will  be  found  that 
most  nerve-cells  have  a  number  of  processes  which  ramify  in 
the  neighbourhood  of  the  cell-body  (dendrons)  and  one  process 
which  does  not  ramify  until  it  has  passed  a  greater  or  less 
distance  from  the  cell-body  (neuron).  The  axis-cylinder  of 
the  medullated  nerve  fibres  is  always  the  neuron  of  a  cell  the 
nucleated  body  of  which  lies  in  a  ganglion  or  other  nerve  centre. 
Since  the  chromate  of  silver  serves  to  stain  nerve-cells  and 
their  processes  and  since  nerve-endings  are  always  the  rami- 
fying terminations  of  nerve-cell  processes  (neurons),  it  is 
natural  to  suppose  that  nerve-endings  will  also  be  stained  by 
this  method.     It  is,  in  fact,  one  of  the  most  valuable  methods 

1  According  to  the  nature  of  the  tissue  and  the  structures  it  is  wished  to 
exhibit.  For  peripheral  endings  of  nerves  6  to  8  days  in  the  osmium-bichro- 
mate mixture  is  best,  and  it  is  often  necessary,  to  get  a  successful  result,  to 
put  the  piece  from  silver  nitrate  back  again  into  osmium-bichromate  for  some 
days,  and  then  again  in  silver  nitrate  (duplicate  process  of  Cajal). 


154  PEACTICAL  HISTOLOaY 

for  displaying  nerve-endings  in  various  parts  (muscles,  glands, 
&c.),  and  is  employed  exactly  as  above  described.  Only  it 
must  be  understood  that  different  organs  require  different 
times  of  immersion  in  the  osmium-bichromate  solution,  and 
the  best  time  for  each  tissue  or  organ  can  only  be  learned  by 
experience.  Previous  hardening  in  formol  does  not  interfere 
with  Golgi's  method. 

Terminations  of  nerves. — The  description  of  the  mode  of 
preparing  and  demonstrating  the  terminations  of  nerve-fibres 
in  various  special  parts  of  the  body  will  be  deferred  until 
those  parts  and  organs  are  severally  treated  of,  but  the  Paci- 
nian bodies,  in  which  many  of  the  sensory  fibres  end,  and  the 
end-plates  in  which  the  nerves  supplying  the  voluntary 
muscles  terminate,  may  be  now  prepared. 

The  Pacinian  bodies  are  very  readily  found  in  the  cat's 
mesentery.  Here  they  are  at  once  seen  when  the  abdomen 
is  opened  and  the  membrane  is  held  up  against  the  light,  as 
clear,  oval  specks,  either  dotted  singly  here  and  there  or 
forming  groups  of  two,  three,  or  more.  There  is  generally  a 
considerable  group  in  the  meso-rectum,  and  moreover  they  are 
here  usually  not  so  much  obscured  by  the  adipose  tissue  as  in 
the  mesentery  proper.  By  far  the  best  general  idea  of  their 
structure  and  the  relation  they  bear  to  the  nerve-fibre  enter- 
ing them  is  obtained  from  their  study  in  the  fresh  condition, 
without  the  addition  of  reagents.  But  it  is  well  to  separate 
them  from  the  surrounding  tissue  of  the  mesentery,  for  this 
is  often  loaded  with  fat,  and,  when  not,  the  fibrous  tissue  of 
the  membrane  tends  to  obscure  the  structure  of  the  little 
bodies. 

In  order  to  isolate  one  of  them  cut  out  the  piece  of  the 
mesentery  containing  it,  carrying  one  of  the  cuts  close  along  the 
edge  of  the  corpuscle.  Then  place  the  excised  piece  upon  a 
slide  in  a  drop  of  serum,  and  withotit  actually  transfixing  the 
Pacinian  itself,  tear  away  the  investing  mesenteric  tissue  bit 
by  bit  under  the  dissecting  microscope.  It  will  be  found  that 
with  a  little  manipulation  the  corpuscle  shells  out  as  a  lemon- 


PACINIAN    CORPUSCLES  155 

shaped  body,  with  a  twisted  stalk  at  one  end.  It  is  to  Ije  ex- 
amined with  a  low  powei"  to  make  sure  that  the  fat  is  entii-ely 
removed  ;  the  debris  may  then  be  wiped  away,  a  little  fresh 
serum  added,  a  narrow  slip  of  thick  paper  placed  close  to  the 
corpuscle  to  prevent  too  great  pressure  of  the  cover-glass,  which 
is  then  laid  on  and  the  specimen  examined. 

In  these  fresh  pi^eparations,  owing  to  the  extreme  trans- 
parency of  the  layers  of  the  capsule,  the  core  of  the  corpuscle 
with  the  central  fibre,  together  with  the  mode  of  passage  of 
the  uerve-tibre  into  this  at  the  stalk,  can  be  made  out  better 
than  after  the  action  of  reagents.  And  still  more  so  if  the 
outer  layers  of  the  capsule  are  removed  altogether,  as  may 
readily  be  done  with  fine  needles  under  the  dissecting  micro- 
scope, leaving  only  the  core  and  the  closely-set  layers  of  the 
capsule  which  immediately  surround  it.  In  tearing  away  the 
outer  layers  it  will  often  happen  that  the  perineural  (Henle's) 
sheath  which  surrounds  the  nerve-fibre  as  this  passes  into  the 
corpuscle  is  torn  off  along  with  them,  since  they  are  directly 
continuous  with  it. 

These  fresh  preparations  of  the  Pacinians  are  very  beauti- 
ful, but  unfortunately  they  cannot  be  preserved  in  that  state. 
Treatment  with  glycerine  causes  the  corpuscles  to  shrink 
and  become  too  transparent,  and  most  of  the  ordinary  staining 
fluids  colour  the  core  too  deeply  and  obscure  the  termination 
of  the  nerve-fibre.  If  it  be  desired  to  preserve  any  such 
preparation  as  showing  some  one  or  more  points  particularly 
well,  the  cautious  employment  of  osmic  acid  prior  to  mounting 
in  glycerine  is  most  to  be  recommended.  The  serum  in  which 
the  corpuscle  is  mounted  must  first  be  replaced  by  salt 
solution  ;  this  again  by  1  per  cent,  solution  of  osmic  acid, 
and  this,  after  being  left  for  an  hour  or  more  in  contact  with 
the  preparation,  by  distilled  water,  whilst  finally  a  drop  of 
glycerine  is  allowed  gradually  to  diffuse  in  from  the  edge  of 
the  cover-glass. 

The  structure  of  the  tunics  which  form  the  lamellated 
capsule  of  the  Pacinian  body  is  not  well  shown  in  the  fresh 


156  PEACTICAL  HISTOLOGY 

preparation  owing  to  the  transparency  of  the  object ;  indeed, 
in  this  it  is  the  lines  of  contact  between  successive  coats  which 
look  like  layers  of  the  capsule  ;  the  substance  of  the  coat, 
being  clear  and  pellucid,  gives  the  notion  of  an  intermediate 
fluid.  In  order  to  show  their  fibrous  connective  tissue  struc- 
ture (the  white  fibrils  running  transversely,  and  collected  for 
the  most  part  near  the  surface  of  each  tunic,  and  the  elastic 
fibrils  forming  a  network  through  the  thickness)  some  of  the 
little  bodies  should  be  dissected  out  in  the  way  above  described, 
and  placed  for  ten  days  or  a  fortnight  in  a  0*2  per  cent,  solution 
of  chromic  acid.  They  are  then  put  up  on  a  slide  in  a  drop  of 
water,  and  with  fine  and  perfectly  clean  needles  are  broken 
up  under  the  dissecting  microscope  bit  by  bit,  commencing 
at  one  end  and  breaking  ofi"  transverse  pieces.  It  will  be 
found  that,  owing  to  the  direction  of  the  fibrils,  the  corpuscles 
tend  to  break  across  into  disc-like  portions.  The  core  does 
not  share  this  tendency,  but  this  is  of  little  consequence 
for  it  is  not  well  displayed  in  these  preparations.  A  piece 
of  hair  having  been  added,  the  preparation  is  covered  and 
examined.  Small  fragments  will  probably  be  found  which 
give  a  sectional  view  of  the  tunics,  and  others  in  which  they 
are  seen  flat.  If  a  little  logwood  solution  is  allowed  to  run 
under  the  cover-glass,  nuclei  on  the  surface  of  the  tunics  may 
here  and  there  be  stained. 

These  nuclei  belong  to  flattened  epithelioid  cells,  which 
cover  both  the  outer  and  inner  surface  of  each  tunic,  and 
which,  seen  in  profile,  are  in  reality  the  well-defined 
lines  seen  in  the  fresh  Pacinian,  and  long  described  as  the 
coats  themselves.  The  outlines  of  these  cells  may  be  brought 
into  view  by  staining  with  nitrate  of  silver.  For  this  purpose, 
as  is  always  the  case  with  silver-preparations,  the  tissue  must 
be  fresh  and  unacted  on  previously  by  any  other  reagent. 
One  or  two  corpuscles  are  to  be  thoroughly  isolated  and 
freed  from  surrounding  mesenteric  tissue  and  fat,  placed  for 
five  minutes  in  silver  solution  (1  in  100),  washed  in  dis- 
tilled water,  and  exposed  in  glycerine  to  the  sunlight  until 


MOTOR   NEKVK-ENDINGS  157 

of   a   greyish  colour,    when  they   may  be    covered,    and    ex- 
amined. 

Sections  of  Pacinian  corpuscles. — To  complete  the  study 
of  the  Paciniaus,  sections  should  be  made  of  them.  A  con- 
venient way  to  prepare  them,  in  order  to  show  the  various 
parts  to  advantage,  is  as  follows  : — A  Aery  small  piece  of 
mesentery  or  meso-rectum  of  the  cat,  containing  several  cor- 
puscles close  together,  is  cut  out  (if  such  can  be  found  ; 
if  not,  one  or  more  may  be  isolated  as  before),  and  placed 
in  a  small  beaker  containing  100  c.c.  of  a  weak  solution  of 
acetic  acid  (1  in  200),  to  which  about  5  c.c.  of  one-half 
per  cent,  chloride  of  gold  solution  has  been  added.  The 
tissue  is  kept  in  this,  in  the  light,  for  tln-ee  or  four  days — 
until  it  has  become  of  a  dark  violet  colour  ;  it  is  then  placed  for 
a  day  in  weak  spirit,  and  then  in  strong,  and  two  or  three 
days  later  is  ready  for  embedding  and  cutting.  In  embedding 
the  piece  of  tissue,  it  should  be  so  placed  that  the  corpuscles, 
at  least  most  of  them,  are  cut  as  nearly  as  possible  trans- 
versely. 

Motor  nerve-endings. — The  end-organs  (end-plates),  or 
terminal  expansions  of  the  motor  nerves,  are  difficult  to  find 
in  fresh  muscle,  and  so  soon  undergo  alteration  as  speedily  to 
become  unrecognisable.  The  best  muscles  to  choose  for  the 
search  are  those  of  a  lamellar  shape  and  with  short  fibres, 
such  as  the  intercostals  of  small  animals.  The  muscular 
fibres  are  severed  close  to  their  attachments,  so  as  to  get  them 
in  their  whole  length,  and  the  small,  thin  piece  of  muscular 
tissue  obtained  is  quickly  transferred  to  a  slide,  and  mounted, 
either  without  addition  of  fluid  or  in  a  small  drop  of  serum, 
which  is  put  on  the  cover-glass  before  this  is  inverted  over  the 
preparation. 

This  should  now  be  thoroughly  searched  with  a  good 
high  power  objective  for  the  nerve-endings.  Branches  of  the 
intercostal  nerve  will  be  found  running  across  the  direction  of 
the  muscular  fibres.  Starting  from  one  of  these,  trace  care- 
fully one  by  one  the  single  nerves  which  pass  off  from  it.     It 


158  PRACTICAL  HISTOLOGY 

will  be  found  generally  that  they  branch  one  or  more  times, 
and  eventually  the  resulting  twigs  pass  'off  to  the  muscular 
fibres,  each  fibre  receiving  one  of  the  nerve-twigs.  They 
retain  their  medullary  sheath  until  the  muscular  fibre  to 
which  they  are  attached  is  reached,  when  the  sheath  suddenly 
ceases  to  be  visible,  and  it  is  by  following  the  single  fibres 
until  they  come  in  this  way  to  an  abrupt  termination,  that  an 
end-organ  may  be  met  with.  But  even  if  the  place  where  the 
nerve-fibre  joins  the  muscular  fibre  is  arrived  at  it  is  still  in 
most  cases  difficult  to  make  out  the  exact  mode  of  termination, 
in  other  words,  the  structure  of  the  organ.  The  utmost 
that  can  generally  be  seen  is  a  clump  of  clear,  round  nuclei 
embedded  in  a  granular  material.  The  difficulty  arises  partly 
from  the  readiness  with  which  these  structures  undergo 
alteration  after  removal  in  warm-blooded  animals,  and  partly 
from  the  fact  that  they  are  often  obscured  by  super-  and  sub- 
jacent muscular  fibres  or  blood-vessels. 

In  the  common  lizard  {Lacerta  agilis)  the  motor  end-organs 
may  be  much  more  easily  found  and  satisfactorily  seen  in  the 
fresh  condition,  but  still  the  utmost  care  must  be  taken  in  the 
preparation.  The  animal  having  been  decapitated  and  the 
trunk  pinned  out  upon  a  cork,  a  piece  of  one  of  the  limb- 
muscles — including  the  whole  length  of  the  fibres — is  removed 
and  placed  on  a  slide  in  a  drop  of  serum.  The  fibres  are  then 
separated  under  the  dissecting  microscope,  carefully  but  not  too 
completely,  and  a  piece  of  paper  or  a  hair  having  been  added 
to  avert  pressure  on  the  tissue,  it  is  covered  and  the  fibres 
are  examined  with  a  high  power  along  their  whole  length. 
If  no  end-organs  can  be  found  in  the  lirst  specimen  another 
must  be  taken,  but  it  will  generally  not  be  very  long  before 
one  is  found,  either  at  the  edge  of  a  fibre,  and  therefore  seen 
in  profile,  or  on  the  surface,  and  seen  flat. 

Permanent  preparations  of  motor  nerve-endings. — Besides 
the  method  of  Golgi  which  has  been  already  given  (p.  152),  two 
other  methods  of  displaying  nerve-endings  may  be  here  described 
as  being  of  gi'eat  practical  value.     They  are  the  methylene  blue 


METHOD   OF   LOWIT  159 

mctliod  of  Elirlich  and  tlie  gold  chloride  method  of  Cuhnheim 
as  modified  by  Lowit.  Of  the  two,  the  former  is  the  more 
general  method,  since  metliylene  blue  appears  to  have  a  specific 
afhnity  for  living  nerve  structures,  and  will  generally,  when  in 
sufficiently  dilute  solution,  stain  these  in  preference  to  any 
other  tissue  elements.  Gold  chloride,  on  the  other  hand,  is  apt 
in  many  tissues  to  stain  the  protoplasm  of  cells  generally,  so 
that  the  nervous  fibrils  which  may  be  also  stained  are  often 
obscured.  Nevertheless  for  displaying  certain  nerve-endings, 
such  as  those  in  muscle  and  in  the  cornea,  there  is  nothinsr 
better  than  a  gold  chloride  preparation.  The  manner  in 
which  these  methods  are  applied  to  show  the  nerve-endings  of 
muscle  may  serve  as  a  type  of  their  application  to  other  tissues, 
although  it  may  be  advisable  in  some  cases  to  adopt  slight 
modifications  in  the  method. 

Application  of  the  gold  chloride  method  to  motor  nerve- 
endings. — A  small  piece  of  muscle  is  taken  from  a  freshly 
killed  animal  (preferably  a  lizard,  from  the  relative  ease  with 
which  the  fibres  may  be  separated  and  the  nerves  followed  to 
their  endings),  and  placed  for  half  to  one  minute  in  a  solution 
of  formic  acid  (1  part  to  4  of  water).  It  is  then  transferred  to 
1  percent,  solution  of  gold  chloride,  and  after  15  minutes  immer- 
sion in  this  it  is  replaced  in  the  formic  solution  and  left  in  this, 
in  the  dark,  for  24  hours,  after  which  it  is  put  into  pure  formic 
acid  for  another  24  hours.  The  tissue  should  then  be  of  a 
dark  violet  colour  on  the  surface  but  redder  near  the  centre. 
Small  shreds  are  torn  off  with  forceps,  placed  on  a  slide  in 
glycerine  and  water,  and  slightly  teased  with  needles,  but  not 
so  as  to  separate  the  individual  fibres.  A  cover-glass  is  now 
placed  on  the  preparation  and  pressure  is  made  upon  it  so  that 
the  tissue  is  flattened  out,  but  without  actually  crushing  the 
muscle-fibres.  The  larger  nerve  branches  are  now  looked 
for  with  a  low  power  and  followed  in  their  ramifications  ;  a 
task  easily  performed  if  the  nerve-fibres  are  stained — as  they 
commonly  are — of  a  dark  violet  colour,  and  the  muscle-fibres 
only  of  a  faint  greyish  violet. 


160  PKACTICAL  HISTOLOGY 

Pieces  of  the  stained  muscle  may  also  be  cut  by  the  freezing 
method.  With  this  object  they  are  placed  first  in  water  to 
remove  the  formic  acid,  then  in  dilute  gum,  frozen,  cut  into 
sections  longitudinal  or  transverse,  and  the  sections  are 
mounted  in  glycerine  or  glycerine -jelly. 

Application  of  the  methylene  hlue  method  to  motor  nerve- 
endings. — Half  a  cubic  centimetre  of  solution  of  methylene  blue 
(1  to  100  of  salt  solution)  is  injected  into  the  anterior  abdominal 
vein  of  a  frog,  and  the  animal  is  killed  after  about  an  hour. 
Small  pieces  of  muscle  (or  of  any  other  tissue  in  which  it  is 
wished  to  show  the  nerve-endings)  are  now  removed,  slightly 
teased  in  salt  solution  and  exposed  to  the  air  on  a  slide  or  in  a 
watch-glass.  After  a  short  time  they  can  be  covered  and 
examined,  when  it  will  be  found  that  the  nerves,  even  in  their 
finest  ramifications,  are  of  an  intense  blue  colour,  the  other 
tissues  being  stained  little  or  not  at  all.  In  the  case  of 
mammals  a  few  cubic  centimetres  of  the  methylene  blue  solu- 
tion is  injected  into  a  vein.  A  modification  of  the  method  is 
to  place  the  tissue  to  be  stained,  after  slightly  teasing  it,  in 
a  dilute  solution  (0-2  per  cent.)  of  methylene  blue  in  salt 
solution  in  a  watch-glass  for  half  an  hour  or  more,  and  then 
proceed  to  examine  it. 

It  has  been  shown  that  the  stain  thus  obtained  by  methy- 
lene blue  can  be  fixed  by  a  proper  application  of  molybdate 
of  ammonia  (Bethe).  To  efiect  this  the  tissue,  after  removal 
from  the  methylene- blue  injected  animal,  or  from  the  methy- 
lene-blue  solution  in  which  it  was  placed,  is  rinsed  with 
normal  saline  and  then  transferred  to  the  following  solution, 
which  should  be  quite  cold  and  freshly  prepared  : 

Molj'bdate  of  ammonia      .         .         .       10  g. 

Distilled  water 100  c.c. 

Hydrochloric  acid       ....       10  drops 

In  this  it  is  left  half  an  hour  to  an  hour  or  more,  accord- 
ing to  size,  thoroughly  washed  with  water,  and  transferred 
to  alcohol  containing  O'S  per  cent,  platinic  chloride  (Cajal). 


METHYLENE    I5LUE   METHOD  161 

After  this  it  can  be  embeckled  and  sections  cut  by  the  usual 
methods. 

If  too  strong  a  inethylene-blue  solution  be  used,  or  it  be 
allowed  to  act  too  long  on  a  tissue,  other  structures  become 
stained  besides  nerve  fibrils,  and  especially  intercellular  sub- 
stance of  connective  tissue  and  epithelia,  so  that  appearances 
like  those  produced  by  silver  nitrate  may  be  obtained. 


162  PEACTICAL  HISTOLOGY 


CHAPTER     VIII 

THE    BLOOD-VESSELS 

The  larger  blood-vessels.— The  epithelial  lining  (endo- 
thelium) can  be  best  demonstrated  in  fresh  blood-vessels  by 
staining  with  nitrate  of  silver.  For  this  purpose  a  piece  of 
a  large  vessel — artery  or  vein — is  obtained,  either  from  a  re- 
cently killed  animal  or  from  an  amputated  limb,  and  having 
been  slit  open  with  scissors  is  pinned  on  a  cork  with  the 
inner  surface  uppermost.  Cai-e  must  be  taken  in  doing  so 
not  to  rub  this  surface  in  any  way.  The  preparation  is  then 
rinsed  with  a  little  distilled  water  from  a  wash-bottle,  with  the 
object  of  removing  any  blood  tliat  may  remain  on  the  wall  of 
the  vessel,  and  a  few  drops  of  half  per  cent,  nitrate  of  silver 
solution  are  allowed  to  flow  over  the  surface.  After  a  minute 
it  is  again  washed  with  distilled  water,  and  is  then  put  at  once 
into  a  beaker  of  spirit  and  placed  in  the  sunlight.  After  a 
time  the  surface  will  have  acquired  a  greyish  tinge,  with  a 
browner  patch  here  and  there  ;  it  may  now  be  removed  from 
the  light,  but  should  be  left  in  spirit  until  the  next  day, 
when  it  will  be  hard  enough  to  enable  thin  sections  to  be 
made  with  a  razor  from  the  inner  surface.  In  order  to  cut 
them  it  will  be  found  convenient  to  remove  the  piece  of 
blood-vessel  from  the  cork,  and  to  hold  it  by  one  end  by 
the  thumb  and  fingers  of  the  left  hand,  so  that  the  piece 
rests  by  its  outer  surface  on  the  ball  of  the  finger  ;  the 
razor  is  then  dipped  into  spirit,  and  as  thin  a  slice  as 
possible  (it  need  not  be  very  large)  is  removed  from  the 
inner  surface  of  the  blood-vessel  and  placed  in  spirit,  after 


BLOOD-VESSELS  1G3 

wliic-h  one  or  two  more  may  be  taken  from  other  parts. 
In  making  sections  when  (he  piece  is  held  in  this  way,  it  will 
be  found  convenient  to  cut /roni  the  operator. 

The  slices  are  taken  upon  a  needle  or  section-lifter,  passed 
through  clove-oil,  and  then  placed  on  a  slide  with  the  stained 
surface  uppermost.  A  drop  of  xylol  balsam  is  added,  and 
then  the  cover-glass.  On  examining  with  the  microscope, 
the  outlines  of  the  epithelioid  cells  will  be  .seen  in  those 
sections  which  were  made  from  ther/rey  part  of  the  blood-vessel. 
In  sections,  however,  which  include  any  of  the  patches  which 
look  broiVN  to  the  naked  eye,  it  will  be  found  that  the  difference 
of  colour  is  due  to  the  epithelial  cells  having  at  these  parts 
become  accidentally  rubbed  or  washed  away  before  the  silver 
solution  was  allowed  to  act  ;  for  since  the  subjacent  tissues  (the 
sub-epithelial  connective  tissue,  if  present,  and  the  muscular 
tissue  of  the  middle  coat)  contaiii  more  intercellular  substance 
than  the  epithelial  layer  (where  it  only  occurs  in  fine  lines  be- 
tween the  cells),  they  assume  a  browner  appearance  after  the 
reduction  of  the  silver,  and  show  under  the  microscope  in 
the  one  case  irregular  wdiite  patches — the  cell-spaces — upon 
the  brown  ground,  and  in  the  other  transversely  arranged 
lanceolate  white  markings — the  plain  muscular  fibre-cells — 
with  a  variable  amount  of  ground-substance  between.  The 
latter  appearance  may  be  obtained  all  over  the  pi'eparation 
if  the  blood-vessel  which  is  to  be  treated  with  silver  nitrate 
is  first  brushed  with  a  camel-hair  pencil  moistened  with  dis- 
tilled water,  for  by  this  means  the  epithelial  cells  are 
removed.  Where  the  sub-epithelial  connective  tissue  is 
absent,  the  elastic  layer  is  the  only  part  of  the  internal 
coat  which  remains,  and  since  this  does  not  reduce  the 
silver  salt  the  muscular  layer  is  the  one  which  is  seen  in 
sucli  cases.  Except  when  the  epithelial  cells  are  first 
removed,  either  purposely  or  accidentally,  even  a  compara- 
tively long  exposure  to  the  action  of  the  nitrate  of  silver 
solution  will  not  cause  the  deeper  coats  to  become  stained. 
This  is  the  case  with  all  structures    whicli  are  coated  with 

m2 


164  PKACTICAL  HISTOLOay 

epithelial  cells,  which  appear  to  resist  the  passage  of  the  silver 
salt  to  the  subjacent  tissues. 

Elastic  layers ;  fenestrated  membrane  ;  muscular  tissue.- 
To  prepare  these  several  parts,  a  piece  of  artery  (or  vein)  is 
taken  (as  fresh  as  possible,  but  this  is  not  so  imperative  as  for 
the  pi-eparation  of  the  epithelial  layer),  and  placed  for  two 
or  three  days  in  a  large  quantity  of  weak  solution  of 
bichromate  of  potash  (about  1  in  800),  or  in  one-third  alcohol. 
The  piece  is  then  taken  out,  pinned  down  on  a  cork,  with  the 
inner  surface  uppermost,  and  a  thin  strip  torn  off  from  the 
inner  surface  with  fine  forceps.  This  is  transferred  to  a  slide, 
and  teased  in  a  drop  of  water.  It  will  be  found  advantageous 
to  employ  only  as  much  water  as  will  keep  the  tissue  moist,  and 
to  add  more  by  placing  a  drop  on  the  cover- glass  before  it  is  laid 
on.  If  the  small  pieces  sire  examined,  it  will  be  found  that  they 
are  for  the  most  part  made  up  of  a  close  network  of  elastic 
fibres  of  varying  degrees  of  fineness.  Many  of  them  have 
very  bi^oad  fibres  and  small  meshes,  so  that  there  may  be 
found  in  difierent  arteries  every  transition  to  the  true 
elastic  fenestrated  membrane.  This  membrane  may  be  met 
with  j)rojecQng  at  the  edges  of  some  of  the  fragments  of 
tissue,  or  even  entirely  separated  ;  the  fragments  are  gene- 
rally curled  at  the  edges,  are  often  striated,  and  nearly 
always  exhibit  rounded  holes.  The  fenestrated  mem- 
branes are  more  frequently  met  with  in  the  inner  coat  of 
some  of  the  smaller  or  medium-sized  arteries  (especially  the 
basilar),  than  in  the  largest, vessels  (such  as  the  aorta). 

But  besides  the  different  kinds  of  elastic  tissue  there  is 
also  to  be  found  in  nearly  every  such  preparation  a  number 
of  plain  muscular  fibre-cells  scattered  about  in  the  fluid  ; 
for,  in  stripping  ofi"  the  inner  coat,  shreds  of  the  middle 
coat  always  adhere  to  it,  and  the  muscular  cells  readily 
separate  after  maceration.  But  the  isolated  cells  present 
in  many  arteries  such  a  ragged  shapeless  aspect  that  they 
would  hardly  be  known  for  muscular  tissue.  A  convincing 
proof,  however,  is  the  addition  of  a  drop  of  weak  hsematein 


BLOOD-VKSSKLS  105 

solution  at  the  edge  of  the  cover-glass.  This,  as  it  comes  to 
each  of  the  cells  in  question,  almost  instantaneously  stains 
their  long  rod-shaped  nuclei  of  an  intense  violet,  whilst  the 
body  of  the  cell,  if  the  logwood  solution  be  sufficiently  weak, 
remains  uncoloured.  The  addition  of  a  little  glycerine  at 
the  edge  of  the  cover-glass,  and  the  subsequent  cementing, 
are  sufficient  to  preserve  the  preparation.  These  prepara- 
tions may  also  be  stained  with  picrocarmine. 

The  connective  tissue  and  elastic  fibres  of  the  outer 
coat  (as  well  as  the  muscular  tissue  of  the  same  coat  in 
certain  veins)  can  also  be  well  seen  in  a  teased-out  pre- 
paration. 

Study  of  the  structure  of  the  blood-vessels  by  means  of 
sections. — To  form  a  correct  idea  of  the  relative  thickness  of 
the  several  coats,  as  well  as  to  observe  the  differences  in 
arrangement  in  different  arteries  and  veins,  it  is  necessary 
to  study  them  in  vertical  sections,  i.e.  sections  made  in  a 
direction  at  right  angles  to  their  surface.  Such  sections  may 
be  either  transverse  or  longitudinal  ;  it  will  be  better  perhaps 
to  choose  the  former  direction,  for  the  middle  coat  is  thereby 
better  displayed.  But  the  vessel  to  be  cut  must  first  be 
hardened.  This  may  be  effected  speedily  by  immersing  it  in 
spirit  for  a  day  or  two,  and  indeed  this  method  can  be 
employed  for  nearly  all  the  tissues  and  organs.  It  is  pre- 
ferable, however,  in  many  instances  to  effect  the  process  more 
slowly,  by  means  of  some  watery  fluid,  such  as  a  3  per  cent, 
solution  of  Ijichromate  of  potash  or  a  0*5  per  cent,  solution 
of  chromic  acid,  since  in  this  way  the  parts  shrink  less  and 
consequently  retain  their  form  better  ;  the  process  should, 
nevertheless,  always  be  completed  by  means  of  spirit.  In 
the  case  of  the  blood-vessels  an  immersion  for  a  fortnight  or 
three  weeks  in  a  3  per  cent,  solution  of  bichromate  of  potash 
answers  well  ;  the  pieces  are  then  placed  for  a  day  in  50  per 
•  cent,  spirit,  and  finally  transferred  to  strong  methylated  alcohol. 
Here  they  may  remain  without  detriment  until  it  is  convenient 
to  px'epare  sections  of  them. 


166  PRACTICAL  HISTOLOGY 

The  smaller  arteries  and  veins  and  the  capillaries. — The 

elongated  epithelial  cells  which  form  the  walls  of  the  capillaries, 
and  which  line  the  arteries  and  veins,  are  readily  shown  in 
silvered  preparations  of  any  vascular  tissue.  It  is  most 
convenient  to  choose  a  vascular  membrane  for  the  purpose, 
because  miore  readily  displayed,  and  of  vascular  membranes 
the  mesentery  of  the  frog  or  toad  is  perhaps  as  easy  to  prepare 
as  another.  The  following  is  the  mode  of  proceeding  : — The 
animal  (a  male)  having  been  decapitated  and  the  spinal  cord 
destroyed,  the  trunk  is  suspended  for  a  few  minutes  by  the 
lower  limbs  in  order  to  allow  the  blood  to  drain  from  the  body 
as  completely  as  possible.  The  frog  is  then  placed  on  its  back 
and  the  abdomen  freely  opened.  A  loop  of  intestine  is  seized 
with  forceps,  drawn  out  and  gently  raised,  while  with  a 
soft  camel-hair  pencil,  moistened  with  distilled  water,  the 
operator  carefully  brushes  the  mesentery  on  both  surfaces, 
carrying  the  brush  in  every  case  froin  the  intestine,  not 
towards  it.  This  brushing  serves  two  purjDOses — in  the 
first  place,  the  epithelial  cells  of  the  serous  membrane,  which 
would  obstruct  the  passage  of  the  silver. solution  to  the  blood- 
vessels, are  removed  ;  and  in  the  second  place,  much  of  the 
blood  which  remains  in  the  vessels  is  driven  out  of  them. 

The  brushing  being  completed,  the  loop  of  intestine, 
with  its  included  mesentery,  is  cut  off,  rinsed  in  a  capsule 
of  distilled  water,  and  at  once  placed  in  1  per  cent, 
nitrate  of  silver  solution.  Here  it  is  allowed  to  remain 
ten  minutes  ;  after  which  it  is  rinsed  in  distilled  water  and 
then  placed  in  common  water  in  the  sunlight.  If  the  day  is 
bright,  the  silver  is  soon  reduced,  and  all  that  remains  to  be 
done  is  to  place  the  preparation  in  a  shallow  glass  dish,  and 
carefully  cut  off  and  remove  the  piece  of  intestine,  leaving  the 
mesentery.  This  must  be  done  under  water,  and  will  require 
sharp  scissors  and  delicate  handling,  so  as  not  to  drag  upon 
the  mesentery  or  throw  it  into  folds. 

In  order  to  mount  it  a  slide  is  held  in  the  water  and  the 
membrane  allowed  to  float  over,  after  which  the  slide  is  care- 


BLOOD-VESSELS  1G7 

fully  lifted  out  with  the  membi'aiie  (lat  upon  it,  and  the  oxccsh 
of  water  is  drained  off.  Before  covering  it  the  preparation 
must  be  examined,  both  with  the  unassisted  eye  and  with  a 
low  power  of  the  microscope,  so  as  to  detect  any  folds  or 
creases  in  it.  If  present  they  can  be  got  rid  of  by  gently 
drawing  out  first  this  corner  and  then  that  with  a  needle. 
A  drop  of  strong  glycerine  may  now  be  placed  on  the  middle 
of  the  preparation,  and  the  co\er-glass  laid  on  and  allowed 
slowly  to  settle  down.  More  glycerine  may  be  afterwards 
added  at  the  edge  if  necessary.  Or,  in  place  of  mounting 
in  glycerine,  the  specimen  may  be  allowed  to  dry  on  the  slide, 
and  may  then  be  mounted  in  xylol  balsam. 

If  there  is  but  little  sunlight  the  reduction  of  the  silver 
may  often  be  better  effected  by  placing  the  loop  of  intestine, 
with  its  attached  mesentery,  after  it  has  been  taken  from  the 
siher  solution  and  rinsed  in  water,  in  a  beaker  of  weak  spirit 
(equal  parts  of  water  and  strong  spirit,  freshly  prepared), 
and  exposing  it  to  the  light  in  this  for  an  hour  or  more.  The 
cutting  off  of  the  intestine  must  be  performed  in  the  same 
fluid,  and  the  mesentery  floated  from  it  on  to  the  slide.  The 
method  has  the  advantage  not  only  of  effecting  the  reduction 
of  the  metal  with  greater  surety,  but  also  of  rendering  it  more 
easy  to  obtain  the  membrane  free  from  creases,  for  the  mesentery 
is  partly  hardened  by  the  spirit  while  in  a  state  of  extension, 
and  continues  in  this  condition  when  floated  on  to  the  slide, 
so  that  it  is  seldom  necessary  further  to  extend  it  by  artificial 
stretching.  In  these  silvered  preparations  little  but  the 
epithelial  cells  can  be  made  out,  for  the  rest  of  the  tissue 
generally  remains  almost  unstained,  and  becomes  yery  trans- 
parent in  glycerine  or  balsam. 

To  exhibit  the  muscular  structure  of  the  small  arteries 
and  veins,  and  the  nuclei  of  their  epithelial  lining  and  of  the 
walls  of  the  capillaries,  the  vessels  are  stained  with  logwood. 
This  is  done  by  immersing  the  mesentery  or  other  vascular 
membrane,  after  having  lain  for  a  few  hours  in  one-third 
alcohol  or  1   in  800  liichromate  of  potash  solution,  in  a  dilute 


168  PEACTICAL   HISTOLOGY 

solution  of  hsematein,  until  distinctly  coloured ;  then  place 
the  tissue  in  water,  and  mount  it,  with  the  same  precaution 
as  before  to  prevent  creasing,  in  glycerine.  For  the  structure 
of  small  arteries  the  pia  mater  from  the  human  brain  may  be 
used.  A  small  piece  is  stripped  off  with  forceps  ;  and  as  it 
consists  almost  entirely  of  small  arteries  and  veins,  and  more- 
over a  few  capillaries  are  generally  dragged  out  with  it  from 
the  cerebral  substance,  the  structure  of  all  these  vessels  is, 
after  staining,  very  well  displayed.  The  small  veins  are  here 
exceptional  in  being  entirely  devoid  of  a  muscular  coat, 
whereas  the  arteries  have  this  coat  well  developed,  and  it  is 
particularly  well  shown  in  consequence  of  the  staining  of  the 
transverse  nuclei  of  the  muscular  fibres  by  the  logwood. 
Within  these  may  be  detected,  by  carefully  using  the  fine 
adjustment  of  the  microscope,  in  the  first  place  longitudinal 
strise,  which  are  produced  by  wrinklings  in  the  elastic  layer  of 
the  internal  coat ;  and  in  the  second  place,  situated  most 
internally,  elongated  oval  nuclei,  which  belong  to  the  epithelial 
cells  lining  the  vessel.  Of  course  two  layers  of  all  these 
structures  are  come  across  in  focussing  from  above  down. 
The  outer  coat  is  represented  merely  by  a  few  corpuscles  and 
fibres  of  connective  tissue  which  blend  externally  with  the 
connective  tissue  framework  of  the  membrane. 

It  will  be  found,  in  carefully  focussing  from  above  down, 
that  at  one  position  of  the  focus  the  small  vessel  has  exactly 
an  appearance  as  if  it  had  been  cut  longitudinally  through 
the  middle,  and  as  if  the  top  of  the  lower  half  were  being 
examined.  The  lumen  is  seen  in  the  centre,  with  possibly  a 
few  blood-corpuscles  still  in  it ;  on  either  side  of  this  a  well- 
marked  line  representing  the  inner  coat ;  outside  this  again 
what  seems  like  a  row  of  rounded  cells,  which  are  really  the 
encircling  fibre- cells  of  the  muscular  coat  seen  as  if  cut  across  ; 
and  finally,  here  and  there,  outside  of  all,  small  cells  pre- 
senting the  fusiform  aspect  of  connective  tissue  corpuscles 
seen  in  profile.  All  these  appearances  are  exactly  the  same 
as  if  a  section  had  been  made  along  the  vessel,  and  result  from 


CIRCULATION  160 

the  fact  that  only  those  parts  of  an  object  which  lie  in  tlie 
horizontal  plane  that  happens  to  coijicide  with  the  focal 
distance  of  the  objective  are  distinctly  seen,  so  that  it  seems 
as  if  only  this  particular  slice  weie  present.  An  'optical 
longitudinal  section '  is  thus  obtained  of  the  vessel. 


STUDY    OF   THE   CIRCULATION 

The  study  of  the  blood-vessels  cannot  be  said  to  be  in  any 
sense  of  the  word  complete  until  they  have  been  viewed  in  the 
living  condition  and  with  the  blood  still  moving  through  them. 
Such  an  observation  can,  of  course,  only  be  made  whilst  an 
animal  is  still  alive,  and  in  parts  which  are  transparent  enough 
to  allow  the  vessels  to  be  distinctly  seen.  Membranous 
parts  are  those  which  are  naturally  best  adapted  for  such 
observation,  as,  for  example,  the  web  of  the  frog's  foot  and 
of  the  bat's  wing,  the  tails  of  tadpoles  or  of  small  fishes  ;  the 
tongue,  mesentery,  and  lungs  of  the  frog  and  toad,  but 
especially  the  latter  animal ;  and  the  mesentery  and  omentum 
of  small  mammals.  In  such  preparations  the  surrounding 
tissues,  and  especially  the  connective  tissue  corpuscles,  may  be 
studied  as  well  as  the  blood-vessels  ;  and  the  changes  due  to 
commencing  inflammation  which  are  exhibited  by  the  blood- 
vessels, and  the  migration  from  the  veins  of  the  white  blood- 
corpuscles,  can  always  be  brought  on  either  by  the  application 
of  irritants  or,  as  in  the  case  of  the  serous  membranes,  by 
simple  exposure  to  the  air.  The  best  methods,  therefoi'e,  of 
observing  the  circulation  of  the  blood  in  diflferent  parts  will 
be  described  in  the  following  preparations. 

Circulation  in  the  frog's  web. — One  of  the  common 
English  frogs  (Eaiui  temj)oraria),  of  as  light  a  colour  as 
possible,  is  placed  in  water  with  which  chloroform  has  been 
shaken  up.  In  this  it  soon  becomes  immobilised,  and  the 
anaesthesia  may  be  easily  kept  up  for  hours  by  keeping  the 
skin  moist  with  the  chloroform  water.  Or  a  few  drops  of  a 
very  weak  solution  of  curare  (1  to  1,000  of  water)  are  injected 


170  PEACTICAL  HISTOLOGY 

under  the  skin  of  the  back.  This  is  generally  sufficient,  in 
the  course  of  from  a  quarter  to  half  an  hour,  to  render  the 
animal  completely  motionless,  whilst  the  pulsations  of  the 
heart  and  the  circulation  proceed  unimpaired.  The  frog  is 
then  laid  on  a  plate  of  glass  of  an  oblong  shape  and  one  of  its 
legs  is  stretched  out  with  the  webs  resting  on  the  glass.  If 
necessary,  the  web  under  examination  may  be  slightly  stretched 
by  being  fastened  on  either  side  by  means  of  threads  attached  to 
the  adjoining  glass  by  modelling  wax.  Care  must  be  taken  that 
the  web  is  not  tightly  stretched,  since  this  tends  to  arrest  or 
obstruct  the  circulation.  A  slip  of  blotting-paper  or  a  piece  of 
linen  rag  is  placed  over  the  animal  and  kept  wetted  with  water, 
and  the  glass,  with  the  frog  upon  it,  is  then  placed  on  the  stage 
of  the  microscope  (the  head  of  the  animal  being  away  from  the 
observer,  and  the  web  over  the  aperture  in  the  diaphragm), 
and  is  fixed  in  this  position  by  the  clamps,  like  an  ordinary 
slide.  To  observe  the  web  a  low  power  is  used  to  see  the 
general  features  of  the  circulation,  a  high  power  being  after- 
wards employed  to  observe  the  parts  more  in  detail.  But  this 
should  not  (unless  it  is  an  immersion)  be  of  too  short  a  focal 
distance,  since  otherwise  the  lower  glass  is  apt  to  become 
clouded  by  moisture  from  the  web.  It  is  not  advisable  to  put 
a  piece  of  covering-glass  on  the  latter  to  prevent  the  clouding, 
as  the  circulation  is  thereby  liable  to  be  interfered  with  or 
arrested.  A  cork  frog-board  with  a  slit  at  one  end  (fig.  51,  a) 
may  be  used  instead  of  a  glass  plate,  and  the  web  gently 
stretched  over  the  slit  by  pin  points. 

The  web  of  the  frog's  foot  is  the  easiest  of  the  vascular 
membranes  to  prepare,  but  has  the  disadvantage  that,  owing 
to  the  thickness  of  its  epidermic  coverings,  it  is  not  always 
easy  clearly  to  make  out  the  intermediate  tissues.  At  the 
same  time,  being  under  almost  completely  natural  conditions, 
the  circulation  will  continue  for  an  indefinite  time  quite  un- 
impaired. 

Circulation  in  the  tails  of  tadpoles  and  fishes. — The  mode  of 
studying  the  circulation  in  these  requires  no  special  description. 


CIRCULATION 


171 


A  tadpole  can  be  readily  iniinobilisod  by  placiiif^  it  in  a  putty  cell, 
throufj;h  a  hole  in  one  side  of  which  the  tail  is  allowed  to  protrude. 
For  small  Hshes  a  special  box  devised  by  Caton  is  used.  The  fish 
is  fixed  scciu'ely  in  this  with  the  tail  projecting,  and  a  stream  of 
water  is  allowed  to  flow  over  the  head  and  gills. 

Circulation  in  the  mesentery. — The  mesentery  of  tlie  frog, 
and  still  better  of  the  toad,  is  admirably  adapted  by  its  thin- 
ness and  perfect  transparency,  as  well  as  its  vascularity,  for 
ol)serv<ations  on  the  blood-vessels  and  surrounding  tissues.     It 


Pic.  rA 


Flat  piece  of  cork  arranged  as  a  frog-stage  for  viewing  the  circulation  in  the 
web,  tongue,  mesentery,  or  lungs.     About  half  natural  size 

Over  the  small  piece  of  cork  a  the  tongue  can  be  fixed  ;  a  can  be  removed  wheu  the  slit 
below  it  is  wanted  for  the  web  ;  li,  cork  \\-ith  a  deep  groove  cut  along  one  side  :  to 
tliis  the  intestine  is  fastened  by  needle  points,  while  the  mesentery  rests  on  a  semi- 
circular piece  of  glass  which  sliould  fit  at  the  top  of  tlie  .sroove 

is  necessary  to  have  a  special  mesentery-plate  for  this  purpose, 
which  can,  however,  be  readily  made  from  an  oblong  piece  of 
cork  or  soft  Avood.  A  I'ound  hole  about  half  or  three-quarters 
of  an  inch  in  diameter  is  made  at  one  side  with  a  cork-borer, 
and  a  small  piece  of  cork  about  half  an  inch  thick,  and  with 
a  segment  of  a  similar  circle  cut  out  of  its  side,  is  fixed  on  the 
board  with  sealing-wax  or  small  pins  (fig.  T)!,  6).  A  piece  of 
glass  of  the  same  shape  as  this  segment  may  be  fitted  into  it 
near  the  top  for  the  mesentery  to  rest  on.  The  animal — pre- 
ferably a  male — having  been  rendered  insensible  by  destruction 


172  PEACTICAL   HISTOLOGY 

of  the  brain,  is  curarised  as  before,  and  laid  upon  its 
back,  and  a  longitudinal  cut  about  an  inch  long  is  made  with 
scissors  through  the  skin  of  the  abdomen,  about  half  an  inch 
to  the  right  of  the  middle  line.  Before  proceeding  further, 
the  operator  should  wait  for  a  minute  or  two  to  make  sure 
that  there  will  be  no  bleeding  ;  and  any  blood  that  may  have 
already  exuded  should  be  dried  up  with  blotting-paper.  The 
abdominal  cavity  is  then  opened  by  a  corresponding  cut 
through  the  muscles  and  peritoneum,  taking  care  to  avoid,  if 
possible,  any  veins  that  may  be  seen.  Having  again  assured 
himself  of  the  absence  of  bleeding,  the  operator  very  gently 
draws  out  one  of  the  coils  of  the  intestine,  with  its  included 
mesentery,  at  the  aperture.  The  animal  is  now  to  be  turned 
over  on  its  side,  and  so  propped  up  against  the  smaller  cork 
that  the  wound  is  about  on  a  level  with  the  top.  All  that 
remains  to  be  done  is  to  place  the  extruded  mesentery  over  the 
aperture,  and,  if  necessary  to  keep  it  in  position,  to  pass 
two  or  three  fine  needle-points  through  the  intestine  into  the 
cork.  In  this  case,  again,  the  greatest  care  must  be  taken  in 
no  way  to  drag  upon  the  exposed  membrane,  nor  to  allow 
it  to  be  pressed  upon.  Moreover,  the  surface  must  from 
time  to  time  be  moistened  with  salt  solution,  to  prevent  its 
becoming  dry.  But  it  will  be  found  that  the  mere  ex- 
posure of  the  serous  surface  to  the  air  is  sufficient  to  produce 
before  long  the  changes  in  the  circulation  (dilatation  of  vessels, 
sticking  and  migration  of  leucocytes)  which  are  characteristic 
of  the  commencement  of  inflammation. 

Capillary  circulation  in  mammals. — It  is  less  easy  to  study  the 
circulation  in  the  serous  membranes  of  mammals,  for  the  exposure 
required  for  the  purpose  is  apt  to  be  far  more  prejudicial  to  the 
maintenance  of  the  normal  condition  of  the  tissues  than  is  the  case 
with  the  cold-blooded  vertebrates.  It  is  necessary,  moreover,  to 
maintam  the  exposed  part  at  the  body  temperature,  and  to  immerse 
it  in  fluid,  siace  it  would  otherwise  become  at  that  temperature 
rapidly  desiccated.  The  membrane  generally  chosen  is  not  the 
mesentery  but  the  omentum,  which  in  many  animals,  e.g.  the 
guinea-pig,  is  very  extensive,  and  at  the  same  time  thm,  and  pro- 


CIRCULATION  173 

vided  in  parts  with  a  sulHcient  number  of  blood-vessels.  Tho 
animal,  which  should  be  rather  a  small  one,  is  ana'sthetised  with 
chloral  hydrate,  one  or  two  cubic  centimetres  of  a  20  per  cent, 
solution  being  injected  under  the  skin.  The  warm  stage  (fig.  J57)  is 
in  the  meanwhile  got  ready,  and  a  glass  tray  (which  can  be  extem- 
porised out  of  a  small  plate  of  glass,  some  pieces  of  glass  rod  and 
sealing-wax)  is  placed  on  it  and  filled  with  0"!)  per  cent,  salt  solution, 
which  is  maintained  at  about  '6ii°  C.  Then  the  animal  is  supported 
on  a  block  at  a  convenient  level,  and  the  abdomen  having  been 
carefully  opened,  a  little  of  the  omentum  is  drawn  out  and  allowed 
to  float  flat  in  the  warm  salt  solution,  where  it  can  be  examined 
either  with  a  low  power  or  with  an  immersion  objective  dipping 
into  the  solution.  If  the  latter  is  employed,  a  piece  of  thin  cover- 
ing-glass must  be  placed  over  the  part  of  the  membrane  which  is 
to  be  examined,  so  as  to  sink  it  in  the  fluid  and  keep  it  steady. 
Biit  in  spite  of  every  precaution,  the  circulation  under  these  condi- 
tions retains  its  normal  character  but  a  short  while,  and  inflamma- 
tory congestion  and  stasis,  or  complete  stoppage  of  the  How  of 
blood,  rapidly  supervenes.' 

Circulation  in  the  lung  of  the  toad. — This  is  readily 
observed  with  the  aid  of  the  mesentery  board.  The  animal 
must,  as  before,  be  first  rendered  insensible  and  curarised  : 
it  will  be  found  that  a  good- sized  toad  will  require  at  least 
six  times  as  much  curare  as  a  frog.  An  opening  is  made  at 
the  side  of  the  chest  large  enough  to  allow  the  lung,  which  in  the 
curarised  toad  almost  always  remains  distended  with  air,  to 
protrude.  The  animal  is  then  propped  up  on  the  mesentery 
board  (fig.  51)  in  such  a  manner  that  the  lung  rests  over  the 
aperture  b,  and  the  circulation  can  be  studied  in  the  part  which 
is  uppermost  without  further  trouble.  A  frog  may  be  used 
in  a  similar  way,  but  there  is  much  greater  difficulty  in  keep- 
ing the  lung  distended.  This  can,  however,  be  done  by  the 
use  of  a  special  apparatus  devised  by  Holmgren.  In  either 
case  the  greatest .  care  nmst  be  takeir  to  avoid  pricking,  or  in 
any  way  ruptui'ing  the  wall  of  tlio  lung. 

^  For  a  detailed  account  of  this  method  tlie  student  is  referred  to  tlie 
original  description  by  Burdon  Sanderson  and  Strieker  in  the  '  Quarterly 
Microscopical  Journal '  for  1870. 


174 


PKACTICAL  HISTOLOGY 


Circulation  in  the  tongue  of  the  toad. — By  far  the  most 

beautiful  object  for  studying  not  only  the  circulation  but  also 
the  tissues  in  the  living  animal,  is  the  tongue  of  the  toad,  and 
in  a  slightly  less  degree  that  of  the  frog.  The  tongue  is  in 
these  creatures  an   extremely  extensile  organ,  which,  under 

Fig.  52 


Structure  and  position  of  the  tongue  of  the  toad  (Dowdeswell) 

A.  Transverse  section  through  the  middle  of  the  organ  with  the  lymph-spaces  fully 

distended ;  a  a,  thick,  papillated  mucous  membrane  ;  6,  thin  lower  membrane  ;  m, 
muscular  bundle  cut  across,  united  to  the  sides  of  the  tongue  by  septa  of  connective 
tissue,  s  s;  V,  position  of  the  larger  blood-vessels 

B.  Profile  view  showing  the  tongue  in  its  ordinary  position  within  the  mouth 

C.  The  same  when  extended 

ordinary  circumstances,  lies  folded  back  on  the  j9oor  of  the 
mouth  (fig.  52,  b),  but  which  can  at  the  will  of  the  animal  be 
protruded  for  a  considerable  distance  (c).  For  the  preparation 
of  the  organ  the  frog-board  is  again  necessary,  a  smaller  piece 
of  cork  of  the  shape  shown  in  the  figure  (fig.  51,  a)  and  about 


CIRCULATION  175 

one-eighth  of  an  inch  thick,  being  fastened  with  pins  over  the 
slit  which  served  for  the  display  of  the  frog's  web  ;  to  its 
aperture  a  thin  piece  of  glass  may  be  adapted. 

The  toad  having  been  I'endered  insensible  and  curarised  as 
before,  is  laid  upon  its  back  with  its  nose  close  to  the  slit. 
Tlie  lower  jaw  is  then  raised  and  the  folded  back  end  of  the 
tongue  is  found,  and  drawn  gently  out  of  the  mouth  with 
forceps.  The  end  has  a  pointed  projection  or  cornu  on  each 
side  ;  these  are  successively  laid  hold  of  by  the  forceps  and 
fastened  with  needle-points  to  the  small  piece  of  cork  on 
either  side  of  the  slit.  Before  the  rest  of  the  operation  is 
described,  a  Avord  or  two  may  be  said  with  regard  to  the 
structure  of  the  organ.  It  is  not  solid  throughout  as  in 
mammals,  but  hollow,  the  interior  being  occupied  by  a 
lymphatic  cavity.  This  lymph- space  is  traversed  by  bundles 
of  muscular  fibres  (fig.  52,  vt)  which  pass  towards  the  ex- 
tremity of  the  organ  and  are  connected  to  the  sides  by 
delicate  septa  of  connective  tissue  (s  s).  Above  the  lymph- 
space  in  the  present  position  of  the  animal — on  its  back  with 
the  tongue  extruded — the  mucous  membrane  is  thick  and 
papillated  (fig.  52,  A,  a  a).  Below  is  a  very  thin  and  delicate 
mucous  membrane  (6),  with  numerous  blood-vessels,  and  small 
muscular  fibres  running  over  it.  The  former  membrane  is 
too  thick  and  irregular  to  allow  the  delicate  internal  struc- 
tures to  be  seen  through  it  ;  it  is  therefore  slit  up  longi- 
tudinally and  pinned  to  either  side.  But  to  do  this  with- 
out injuring  the  parts  below,  it  must  be  well  separated 
from  them,  and  this  can  best  be  effected  by  distending  the 
lymph-sac  with  salt  solution.  With  this  object  a  hypo- 
dermic syringe,  provided  with  a  fine  and  sharp  cannula,  is 
filled  with  the  fluid,  and  its  point  is  stuck  into  the  tongue 
near  the  end,  passing  about  half  an  inch  backwards.  It  will 
almost  certainly  be  found  that  on  pressing  the  piston  down 
the  salt  solution  will  readily  flow  into  the  lymph-sac  and,  as 
it  fills  this,  will  cause  the  thin  mucous  membrane  at  the  lower 
part  to  become  bagged  out  and  completely  separated  from  the 


176  PRACTICAL  HISTOLOGY 

muscular  bundles,  m  m,  and  these  again  from  the  thick  layer 
above.  The  latter  is  now  carefully  slit  up  along  its  middle  by 
sharp  fine  scissors,  and  first  one  edge  of  the  wound  and  then 
the  other  is  drawn  to  the  side  of  the  slit  in  the  cork  and 
fastened  there  by  two  or  three  needle-points.  If  everything 
is  carefully  done  there  will  be  no  escajje  of  blood  over  the 
preparation ;  but  should  any  blood  have  exuded  it  may  be 
washed  off  by  pouring  a  little  salt  solution  over  the  surface. 

There  is  now  brought  to  view  the  fan-like  group  of  muscles 
which  pass  through  the  middle  of  the  lymph-sac,  and  the 
bundles  of  v/hich  are,  as  before  mentioned,  connected  with 
the  sides  and  with  one  another  by  delicate  septa  of  connective 
tissue,  traversed  by  a  few  blood-vessels  ;  and  it  is  this  delicate 
connective  tissue,  of  which  two  strata  can  generally  be  traced, 
one  superficial  to  the  other,  which  is  best  adapted  for 
exhibiting  both  vessels  and  connective  tissue  corpuscles. 
Moreover,  the  mere  exposure  of  the  lymphatic  surface  soon 
causes  inflammatory  changes,  and  after  the  preparation  has 
been  made  a  few  minutes  only,  the  commencement  of  these 
is  seen  iri  the  sticking  of  the  pale  corpuscles  to  the  walls  of 
the  vessels,  speedily  followed  by  their  migration  from  the  veins 
into  the  surrounding  tissue.  Nowhere  can  the  fact  be  more 
clearly  established,  and  the  details  of  the  process  moi'e  accu- 
rately followed  than  here.  The  circulation  of  the  blood 
among  the  muscular  fibres  can  also  be  well  seen  in  this  part  of 
the  tongue. 

Lastly,  by  focussing  through  the  connective  tissue  septa, 
or  by  severing  the  longitudinal  muscular  bundles  which  they 
serve  to  unite,  the  vessels  of  the  lower  mucous  membrane  are 
brought  into  view,  especially  if  a  slip  of  glass  is  fitted  into  the 
small  piece  of  cork,  so  as  to  support  the  tongue  and  prevent 
the  thin  membrane  from  bulging  downwards. 

METHOD    OP    INJECTING    THE    BLOOD-VESSELS 

Before  leaving  the  subject  of  the  blood-vessels  the  best 
mode    of  filling    them    with    transparent    material    may    be 


:\IETIIOI),S   OF   INJECTING  177 

described,  especiidly  as  in  the  study  of  the  several  organs  it 
is  necessary,  in  order  that  the  course  and  arrangement  of 
the  vessels  may  be  properly  made  out,  that  sections  of  in- 
jected as  well  as  of  uninjected  preparations  should  be  looked 
at.  Tt  will  be  convenient  in  this  place  to  describe  the  injec- 
tion of  a  .small  animal  entire  from  the  aorta,  reserving  any 
special  directions  concerning  organs  which  are  not  thereby 
properly  injected,  such,  for  instance,  as  the  lungs  and  liver, 
until  they  are  severally  dealt  with. 

Preparation  of  the  injection  mass. — Tliis  is  almost  always 
a  solution  of  gelatine  coloured  either  red  with  finely  precipi- 
tated carmine,  or  blue  with  soluble  Prussian  blue.  Some- 
times, but  rarely,  when  it  is  wished  to  inject  two  sets  of  vessels 
of  different  colours,  both  of  these  are  used,  but  as  a  rule  all 
the  blood-vessels — arteries,  capillaries,  and  veins — are  filled 
Avith  the  same  injecting  fluid  ;  preparations  in  which  the 
arteries  are  filled  with  one  colour  and  the  veins  with  another 
are  difficult  to  prepare,  and  pi*esent  no  practical  advantage. 
The  gelatine  solution  is  made  as  follows  : — Ten  grammes  of 
clear  gelatine  cut  into  small  pieces  is  placed  in  a  beaker 
containing  50  c.c.  of  cold  distilled  water.  In  an  hour  or  two 
the  gelatine  will  have  swollen  to  several  times  its  original 
volume.  A  glass  cover  is  put  over  the  beaker,  and  it  is  placed 
in  a  water-bath  and  heated  until  the  gelatine  is  rendered  fluid. 

For  the  red  injection  4  grammes  of  carminate  of  ammonia 
is  rubbed  up  in  a  mortar  with  50  cubic  centimetres  of  dis- 
tilled water.  When  the  carminate  of  ammonia  is  as  completely 
as  possible  dissolved  the  liquid  is  filtered  and  the  filtrate  is 
warmed.  The  gelatine  solution  is  then  gradually  added  to  it 
with  constant  stirring.  The  next  part  of  the  process  is  to 
precipitate  the  carmine,  for  otherwise  it  would  difl'use 
through  the  walls  of  the  vessels  and  colour  the  tissues  ;  but 
it  must  be  precipitated  so  finely  that  the  particles  shall 
not  be  visible  even  under  the  highest  power  of  the  microscope. 
To  efifect  this  object  the  solution  is  to  be  acidulated  by  running 
a  small  quantity  of  10  per  cent,  solution  of  acetic  acid  from 

N 


178  PRACTICAL   HISTOLOGY 

a  burette  drop  by  drop  into  the  warm  carminised  gelatine  solu- 
tion, which  is  all  the  while  constantly  agitated.  The  alteration 
in  reaction  may  be  shown,  in  spite  of  the  red  colour  of  the 
solution,  by  placing  a  small  drop  on  the  hack  of  a  piece  of 
glazed  blue  litmus  paper  ;  if  the  coloured  face  of  the  paper  be 
looked  at  it  will  be  found  to  have  assumed  the  characteristic 
red  which  acids  produce,  and  which  is  quite  different  from 
carmine.  This  change  is  caused  by  the  diffusion  of  the  acetic 
acid  through  the  paper,  whereas  the  carminised  gelatine  sets 
almost  immediately,  and  is  thus  unable  to  soak  through.  Or  a 
drop  of  the  injection-mass  to  be  tested  may  be  put  on  the  glazed 
coloured  face  of  the  litmus  paper,  and  after  half  a  minute  wiped 
off  with  a  piece  of  linen  wetted  with  distilled  water.  The 
spot  where  it  had  rested  will,  if  it  be  acid,  show  characteristic 
change  in  colour. 

It  is  not  sufficient  that  the  fluid  should  only  just  be  acidu- 
lated ;  there  must  be  an  excess  of  acid.  A  few  more  drops 
are  therefore  added,  and  the  carmine  completely  thrown  out 
of  solution.  This  change  from  the  soluble  to  the  insoluble 
state  is  accompanied  by  a  marked  alteration  in  colour,  for 
whereas  whilst  still  in  solution  the  carmine  imparted  the 
rich  deep  red  of  an  ammoniacal  solution  to  the  gelatine, 
after  the  precipitation  the  colour  of  the  latter  changes  to  a 
paler  red,  comparable  rather  to  the  tint  presented  by  the 
powdered  carmine  in  the  dry  state.  Even  after  the  pro- 
duction of  this  change  a  few  more  drops  of  the  acetic  acid 
may  be  added,  for  it  will  do  no  harm,  and  will  tend  to  counteract 
the  natural  alkalinity  of  the  tissues. 

The  coloured  gelatine  is  next  strained  through  a  piece 
of  flannel  or  fine  linen,  previously  soaked  in  hot  water  and 
again  wrung  out,  and  is  collected  in  a  flask  as  it  runs  through 
the  filter,  and  transferred  to  the  injecting-bottle. 

Por  the  blue  injection  10  grammes  of  gelatine  is  taken, 
and  after  having  been  soaked  in  cold  water  and  dissolved 
up  as  before,  50  c.c.  of  a  2  per  cent,  solution  of  Berlin 
blue,  which  has  been  previously  warmed,  is  gradually  added 


METHODS   OF   INJECTING  179 

with  constant  agitation  to  the  fluid  gelatine.  The  blue  mix- 
ture is  strained,  and  is  then  ready  for  use,  without  the 
necessity  of  precipitating  the  colouring  matter,  for  this 
being  colloid  does  not  diffuse  readily. 

It  is  sometimes  advantageous  in  cases  where  the  structure 
of  the  walls  of  the  blood-vessels  is  to  be  the  subject  of  observa- 
tion, to  use  an  injection-mass  which  is  far  less  deeply  coloured. 
This  can  of  course  be  readily  obtained  by  diminishing  the 
proportion  of  carmine  or  Berlin  blue  which  is  used. 

The  soluble  Berlin  blue  is  of  great  value  for  the  purpose  of 
injecting  both  the  blood-vessels  and  lymphatics.  It  is  somewhat 
troublesome  to  prepare.  The  following  is  the  method  recommended 
by  Briicke,  to  whom  we  owe  its  introduction : — 

Take  of  potassic  ferrocyanide  217  grammes,  and  dissolve  in  a 
litre  of  water  (solution  a). 

Take  a  htre  of  a  10  per  cent,  solution  of  ferric  chloride  (solu- 
tion b). 

Take  four  litres  of  a  satm-ated  solution  of  sulphate  of  soda 
(solution  c). 

Add  A  and  b  each  to  two  litres  of  c.  Then  with  constant  stirring 
pour  the  ferric  chloride  mixture  into  the  ferrocyanide.  Collect  the 
precipitate  upon  a  flannel  strainer,  returning  any  blue  fluid  which 
at  first  escapes  through  the  pores  of  the  flannel ;  allow  the  solutions 
to  drain  off;  pour  a  little  distilled  water  very  carefully  over  the 
blue  mass,  retm-ning  the  first  washings  if  coloured,  and  renew  the 
water  from  day  to  day  until  it  drips  through  permanently  of  a  deep 
blue  colour.  This  is  a  sign  that  the  salts  are  washed  away,  and 
all  that  is  further  necessary  is  to  collect  the  pasty  mass  from  the 
strainer  and  allow  it  to  dry. 

Apparatus  employed  for  injecting. — This  consists,  in  the 
first  place,  of  a  bottle  for  holding  the  coloured  fluid  ;  and 
secondly,  of  some  means  of  producing  a  steady,  elastic,  and 
readily  alterable  pressure  on  the  surface  of  the  fluid  so  that  it 
may  be  driven  with  any  required  force  into  the  arteries.  Fig. 
53  represents  a  convenient  form  of  apparatus  for  general  use. 
The  bottle  («),'  which  holds  the  injecting  fluid,  is  a  moderate- 

'  A  similar  one  is  shown  on  a  larger  scale  in  fig.  5G,  at  c. 

n2 


180 


PEACTICAL  HISTOLOGY 


sized,    wide-mouthed    phial,    with    a    well-fitting   vulcanised 
indiarubber    cork,    through   which    three    glass   tubes    pass. 


Fig. 


Injecting  apparatus.     Complete 

«,  condensing  syringe,  iixed  to  the  table ;  p,  pressure  bottle ;  6,  beaker  of  warm  water  in 
which  the  injection-bottle,  i,  stands  ;  V,  small  beaker  containing  salt  solution  ;  w, 
water-bath  heated  by  a  ring  burner  below  :  the  temperature  of  the  water  is  indicated 
by  a  thermometer,  t,  placed  in  it ;  c,  arterial  cannula,  connected  to  an  indiarubber 
tube  from  the  injecting-bottle  ;  close  to  the  cannula  is  a  steel-clip.  The  cannula  rests 
upon  a  glass  ijlate,  whioli  may  serve  either  to  put  the  animal  which  is  being  injected 
upon,  or  to  cover  it  over,  if  it  is  thought  necessary  to  place  it  in  the  water-bath 

One  of  these  goes  to  the  bottom,  and  from  it  an  indiarubber 
tube  passes,  which  will  be  subsequently  connected  with  the 
artery  cannula  (c),  but  not  before  this  has  been  inserted  in  the 


METHODS   OF  INJECTING  181 

blood-vessel,  in  the  manner  immediately  to  Vje  described.  Tha 
other  passes  only  just  through  the  cork,  and  serves  to  maintain 
communication  by  means  of  another  indiarubber  tube  with  the 
pressure-bottle  p.  The  third  tube  is  connected  with  a  pres- 
sure-gauge, which  may  be  a  small  mercurial  manometer. 
The  injection-bottle  is  placed  during  the  process  of  injecting 
in  a  glass  vessel  (6)  of  warm  water  (about  40°  C.)  ;  a  piece 
of  cork  is  wedged  in  betweeii  the  bottle  and  the  side  of 
the  vessel  to  prevent  the  bottle  from  floating  up  as  it 
becomes  emptied  of  injection,  and  the  vessel  is  covered  with 
a  glass  plate  (not  shown  in  the  figure).  The  pressure- 
bottle  is  a  large  glass  or  earthenware  bottle  of  from  one 
to  three  gallons  capacity,  and  tightly  fitted  with  an  india- 
rubber  cork,  through  which  two  glass  tubes  pass.  One  of 
these  is  connected,  as  before  mentioned,  with  the  injection- 
bottle,  and  the  other  with  a  condensing  syringe  (s),  by  means 
of  which  the  air  within  the  bottle  can  be  brought  to  any 
state  of  tension  that  may  be  desired.  Finally,  if  the  injec- 
tion is  to  occupy  a  considerable  time,  a  water-bath  («c),  heated 
by  a  ring-bui'ner  to  about  40°  C,  should  be  provided  for 
receiving  both  the  beaker  containing  the  injection-bottle  and 
the  animal,  and  maintaining  their  temperature  during  the 
process.  Ordinarily,  however,  if  the  operation  be  quickly  and 
dexterously  performed,  the  whole  process  will  not  occupy 
more  than  a  few  minutes,  and  will  be  over  before  the  natural 
heat  of  the  body  has  had  time  to  become  dissipated. 

Everything  then  being  in  readiness,  the  animal — a  rabbit, 
guinea-pig,  or  rat,  for  example— is  killed  by  chloroform 
inhalation,  being  placed  under  a  bell-glass  with  a  sponge 
wetted  with  chloroform.  As  soon  as  it  is  dead  it  is  taken  out 
and  held  by  an  assistant,  whilst  the  operator  first  quickly 
reflects  the  skin  from  the  front  of  the  thorax  and  then  makes 
an  opening  in  that  cavity  just  over  the  position  of  the  heart. 
This  is  then  seized  near  the  apex  with  blunt  forceps,  drawn 
out  of  the  aperture,  and  held  here  by  an  assistant.  The 
aorta  is  then  found,  the  point  of  a  pair  of  forceps  passed  under 


182  PEACTICAL  :^ISTOLOGY 

it  close  to  the  heart,  and  a  thread  ligature  drawn  round  it. 
A  snip  is  now  made  in  the  left  ventricle,  and  an  arterial 
cannula  (fig.  54  c.',  c.^,  c.^)  passed  through  this  into  the  aorta, 
in  which  it  is  tied  by  the  ligature.  Then  by  means  of  a  pipette 
a  little  warm  water  or  salt  solution  is  passed  into  the  cannula 
so  as  completely  to  fill  it  to  the  exclusion  of  air. 

The  next  thing  to  do  is  to  connect  the  cannula  with  the 

Pig  54 


Cannulas  for  injecting.     Natural  size 

c.\  c?,  c.',  glass  cannulas  of  different  sizes ;  C",  metal  oanniila ;  it  is  sometimes  more  easy 
to  insert  than  the  glass  ones,  especially  into  fine  blood-vessels,  or  into  lymphatics. 
fZ,  steel  clip  for  clamping  an  artery,  or  a  small  iudiarubber  tube,  a  anil  6  are 
intended  to  illustrate  the  mode  of  making  the  glass  cannulas  ;  a,  glass  tube  which 
has  been  heated  in  the  middle  in  the  blow-pipe  flame,  and  drawn  out  so  as  to  be 
narrower  here  ;  6,  the  same  tube  after  having  been  again  heated  (by  the  tip  of  the 
flame),  and  drawn  out  at  the  points  x  x,  so  as  to  narrow  it  still  more  at  those 
places.  The  subsequent  proceeding  consists  in  making  a  nick  at  I  with  the  edge  of 
a  file,  breaking  the  tube  across  here,  and  with  a  fine,  flat,  wetted  file  grinding  the 
ends  away  obliquely  as  far  as  the  dotted  ring  in  each.  The  sharpness  of  the  filed 
edge  is  got  rid  of  by  inserting  it  for  a  moment  or  two  in  the  flame.  Two  similar 
cannulas  are  thus  made  from  one  piece  of  tubing 

indiarubber  tube  which  brings  the  injection  from  the  bottle. 
But  this  tube  must  first  be  completely  filled  by  the  injection, 
so  that  it  contains  no  bubble  of  air.  To  effect  this,  whilst  the 
indiarubber  tube  is  kept  closed  by  the  spring  clip  with 
which  it  is  provided,  the  air  in  the  apparatus  is  put  under 
a  pressure  of  about  two  inches  of  mercury  by  working  the 
syringe.  The  free  end  of  the  indiarubber  tube  is  now  held 
up,  and  the  clip  opened  until  the  coloured  fluid  forced  up 
by  the  pressure  begins  to  escape,  when  the  clip  is  immediately 


METHODS   OF  INJECTING  183 

■closed,  and  the  tube  is  slipped  on  to  the  arterial  cannula. 
The  greatest  care  must  be  taken  throughout  to  avoid  thn 
introduction  of  air,  since  this  would  obstruct  the  smaller 
vessels  and  prove  fatal  to  the  success  of  the  injection. 

The  clip  is  now  permanently  opened  and  the  injection 
suffered  to  How  into  the  aorta,  at  first  under  the  low  pressure 
of  two  inches  of  mercury  ;  but  the  pressure  is  gradually  in- 
creased by  working  the  syringe  until  a  pressure  of  three  or  four 
inches  is  attained.  The  blood  in  the  vessels  gets  forced  before 
the  injection  into  the  right  cavities  of  the  heart,  so  that  these 
are  soon  much  distended  ;  when  this  is  the  case  the  nght 
ventricle  is  slit  open  and  the  accumulated  blood  allowed  to 
flow  out.  The  blood  is  soon  followed  at  first  by  a  mixture  of 
blood  and  the  coloured  gelatine,  but  afterwards  by  the  latter 
only  ;  after  this  has  been  escaping  for  a  minute  or  two,  the 
slit  in  the  ventricle  is  closed  by  placing  a  clip  on,  or  tying  a 
tape  round  the  heart,  and  the  injection,  being  thus  obstructed 
in  its  outflow,  accumulates  in  the  vascular  system  and  dis- 
tends all  the  vessels  to  their  fullest  extent.  The  success  of  the 
injection  may  be  estimated  by  the  colour  of  those  parts  which 
are  not  concealed  by  the  fur — the  paws,  lips,  nose,  and  ears, 
for  instance — and  the  tongue  and  interior  of  the  mouth.  After 
waiting  a  few  minutes  longer,  until  the  vessels  may  be  con- 
sidered to  be  completely  filled,  a  tape  ligature  is  put  round 
the  base  of  the  heart,  so  as  to  include  all  the  great  blood- 
vessels, and  is  slowly  tightened.  This  will  effectually  prevent 
any  escape  of  the  fluid  gelatine  from  the  vessels  when  the 
cannula  is  removed  from  the  aorta.  This  may  therefore  now  be 
effected,  the  clip  having  been  first  replaced  on  the  indiarubber 
tube  which  is  connected  with  it,  so  as  to  prevent  the  injection 
from  spurting  out. 

The  animal  is  then  put  aside  until  it  has  become 
cold  and  the  gelatine  is  fii-mly  set.  Any  parts  which  are 
wanted  are  then  cut  out  and  placed  either  in  60  per  cent, 
spirit,  to  be  afterwards  gradually  transferred  to  strong  spirit, 
or  they  may  with  advantage  be  placed  in  3  per  cent,  solution 


184  PRACTICAL  HISTOLOaY 

of  bichromate  of  potash,  in  which  they  remain  for  a  fortnight 
or  longer,  or  in  10  per  cent,  formol. 

The  blue  injection  possesses  the  disadvantage  that  in 
spirit  it  is  apt  to  become  temporarily  reduced  in  the  smaller 
vessels  and  rendered  almost  colourless,  so  that  it  is  difficult 
to  determine  whether  a  successful  injection  has  been  made- 
or  not.  The  colour  may,  however,  be  readily  restored  by 
pouring  some  oxidising  fluid,  such  as  spirits  of  turpentine  or  a 
weak  solution  of  peroxide  of  hydrogen,  over  any  part  about 
which  there  is  doubt ;  and  in  the  ordinary  course  of  preparing 
and  mounting  sections  the  blue  colour  is  always  brought  back, 
especially  if  turpentine  is  used  as  a  clearing  fluid. 

The  preparations  in  bichromate  may  conveniently  be  cut 
by  the  freezing  method. 

Most  other  forms  of  apparatus  which  are  used  for  injecting^ 
are  more  or  less  like  the  one  above  described,  the  chief  modi- 
fication being  in  the  mode  in  which  the  pressure  is  produced,, 
this  being  effected  in  one  form  by  allowing  water  to  flow  from 
a  tap  into  the  pressure- bottle  (which  in  such  cases  is  generally 
made  of  metal),  and  thus  compressing  the  air  ;  in  another  by 
allowing  mercury  to  flow  from  one  vessel  into  another.  But 
the  latter  method,  although  useful  when  small  quantities  of 
injecting-fluid  only  are  required,  as  with  the  injection  of  the 
lymphatics  (with  which  the  apparatus  will  be  described),  is 
costly  for  large  quantities  ;  and  it  will  be  found  that  none  are 
more  simple  and  efficient  in  working  than  the  one  here  recom- 
mended. 

If  a  condensing  syringe  is  not  at  hand,  sufficient  pressure 
may  be  got  in  many  cases  merely  by  hloioing  air  into  a 
pressure-bottle  through  an  indiarubber  tube,  its  escape  being 
prevented  by  subsequently  clipping  the  tube. 

Injections  which  are  fluid  in  the  cold  (of  which  the  best  is 
a  1  or  2  per  cent,  solution  of  Berlin  blue)  are  sometimes 
used  for  the  blood-vessels,  especially  for  injecting  cold-blooded 
animals. 

Any  of  the  coloured  gelatine  that  may  remain  over  can  be 


METHODS   OF  INJECTIxN'G  185 

preserved  until  .again  wanted,  if  the  precaution  is  taken,  after 
disconnecting  from  the  pressure-bottle  and  allowing  the  fluid 
in  the  cannula  tuhe  to  run  back,  to  place  the  bottle,  tubes  and 
all,  for  twenty  minutes  in  boiling  water,  and  whilst  still  hot  to 
stopper  up  the  ends  of  the  tubes  with  pieces  of  glass  rod.  The 
whole  can  then  be  put  away  until  wanted. 

If  it  is  desired  to  show  the  endothelium  of  the  .small  blood- 
vessels the  silver-gelatine  mass  which  is  recommended  on  p.  210 
for  showing  the  epithelium  of  the  pulmonary  alveoli  may  be 
employed,  or  simply  a  solution  of  silver  nitrate  of  1  per  1,000 
in  distilled  water  may  be  used.  But  in  either  case  the  vessels 
should  first  be  washed  out  with  a  2  per  cent,  solution  of  nitrate 
of  soda  in  distilled  water. 


186  PRACTICAL  HISTOLOGY 


CHAPTER   IX 

LYMPHATICS.      SYNOVIAL    MEMBRANES.       SEROUS   MEMBRANES 

It  is  in  preparations  of  the  serous  membranes  that  the  struc- 
ture and  arrangement  of  the  lymphatic  vessels  can  be  best 
demonstrated,  and  it  will  on  this  account  be  convenient  to 
combine  them  here  under  one  head,  especially  as  the  method 
which  on  the  whole  exhibits  the  structure  of  the  serous  mem- 
branes best  is  the  only  one  which  shows  at  all  satisfactorily 
the  structure  of  the  lymphatic  vessels  and  their  relation  to 
the  cell-spaces  of  the  connective  tissue. 

Preparation  of  the  rabbit's  omentnin  by  the  silver  method. 
A  rabbit  (half-grown)  having  been  killed  by  bleeding,  the 
abdomen  is  opened,  and  the  omentum,  which  is  generally  to 
be  found  crumpled  up  close  beneatli  or  to  the  left  of  the 
stomach,  is  raised  with  forceps,  cut  off  as  close  to  the  line  of 
attachment  to  the  stomach  as  possible,  and  placed  in  a  shallow 
dish  of  2  per  cent,  solution  of  nitrate  of  soda  in  distilled  water 
which  is  at  hand  to  receive  it.  Besides  this  solution  there 
should  be  ready  on  the  table  a  little  1  per  cent,  solution  of 
bichromate  of  potash,  some  one-half  per  cent,  solution  of 
nitrate  of  silver,  a  wash-bottle  of  distilled  water,  a  flat  glass 
dish  containing  a  mixture  of  spirit  and  water  (equal  parts), 
two  glass  plates  about  four  inches  by  six,  a  large  soft 
camel-hair  brush,  and  two  or  three  clean  capsules  and  watch- 
glasses. 

Two  small  corners  are  first  to  be  cut  off  the  omentum. 
One  of  these  is  placed  in  the  bichromate  of  potash,  put 
aside,    and   examined   after   two   or  three  days  ;  this  is  for 


LYMPUATICS  187 

exhibiting  the  arrangement  of  the  connective  tissue  Hl^res. 
The  other  is  first  rinsed  with  nitrate  of  soda  solution,  then 
placed  for  one  minute  in  a  watch-glass  containing  a  little  of 
the  silver  solution,  rinsed  again  with  distilled  water,  and  ex- 
posed to  the  sunlight  in  another  watch-glass  containing  water. 
After  from  a  few  minutes  to  half  an  hour  of  exposure,  accord- 
ing to  the  intensity  of  the  light,  it  may  be  removed,  and  a 
portion  or  the  whole  of  it  cautiously  mounted  by  being  floated 
upon  a  slide  under  water.  The  excess  of  water  is  removed 
from  the  slide,  all  creases  and  folds  are  carefully  got  rid  of  in 
the  same  way  as  with  the  frog's  mesentery,  before  described, 
and  finally  either  a  drop  of  glycerine  is  added  and  the  cover- 
glass  superposed,  or  the  preparation  is  dried  and  mounted  in 
xylol  balsam.  This  preparation  is  for  the  purpose  of  show- 
ing the  epithelial  layer  which  covers  each  surface  of  the 
membrane.  If  only  a  portion  were  mounted,  the  rest  may  be 
placed  for  a  few  minutes  in  hpemalum  before  mounting  ;  in 
this  way  the  nuclei  of  the  cells  may  be  brought  to  view. 

But  while  this  second  corner  was  being  exposed  to  the 
light  the  preparation  of  the  rest  of  the  omentum  can  be  pro- 
ceeded with. 

In  the  first  place,  it  is  floated  on  to  one  of  the  glass  plates 
and  removed  from  the  fluid,  and  then  by  drawing  gently  first 
at  one  place  and  then  at  another  the  creases  and  folds  are 
gradually  removed,  and  it  is  in  this  way  spread  out  as  an 
exquisitely  delicate  membrane,  which  may  be  made  to  cover 
the  whole  upper  surface  of  the  glass  plate,  and  may  be  ex- 
tended round  its  edges  so  as  to  reach  the  lower  surface.  As 
soon  as  all  folds  are  in  this  way  removed  from  the  part  which 
covers  the  upper  surface  of  the  plate,  the  second  glass  plate  is 
applied  to  the  under  surface  of  the  first,  and  the  membrane, 
or  at  any  i-ate  its  greater  part,  in  thus  maintained  in  an  ex- 
tended state.  The  two  glass  plates  can  then  be  fixed  together 
by  indiarubber  bands.  Next  the  surface  is  gently  brushed  all 
over  with  the  camel-hair  pencil  moistened  with  nitrate  of  soda 
solution  ;  this  is  for  the  purpose  of  removing  the  epithelial 


188  PEACTICAL  HISTOLOGY 

layer  from  that  surface,  and  enabling  the  silver  solution  more 
rapidly  to  penetrate.  The  brushing  is  not  absolutely  essential, 
for  in  many  parts,  especially  those  in  which  the  lymphatics 
and  blood-vessels  are  most  numerous,  the  epithelial  layer  is 
deficient,  or  at  least  incomplete  and  modified. 

The  solution  is  now  drained  ofi^  the  membrane  as  much 
as  possible,  and  without  delay  a  quantity  of  nitrate  of  silver 
solution  is  poured  on  it  and  allowed  to  run  over  every  part 
of  the  exposed  surface.  After  five  minutes  it  is  washed  away 
with  distilled  water,  and  the  glass  plates,  with  the  membrane 
of  course  still  upon  the  stirface  of  the  upper  one,  are  placed 
in  50  per  cent,  spirit  contained  in  a  flat  glass  dish.  The  dish 
is  then  covered  and  placed  in  the  sunlight  until  the  silver,  as 
evidenced .  by  the  change  in  colour,  is  fully  reduced.  Small 
pieces,  each  about  an  inch  square,  are  then  cut  with  sharp 
scissors  from  various  parts,  floated  on  to  slides  with  the 
browned  surface  uppermost,  and  are  exposed  to  the  air  for 
a  few  minutes  to  allow  most  of  the  S2nrit  to  evaporate,  leav- 
ing them  in  water  ;  to  this  a  drop  of  glycerine  is  added,  and 
finally  the  cover-glass  is  superposed.  Or  the  preparation  is 
allowed  to  dry  on  to  the  slide  and  is  then  simply  mounted  in 
xylol  balsam.  Before  mounting  in  glycerine,  the  cell-nuclei 
may  be  stained  by  hsematoxylin  after  the  silver. 

Both  before  and  after  the  treatment  with  nitrate  of  silver 
it  may  have  been  noticed  that  the  delicate  membrane  is 
studded  all  over  with  patches  of  thicker  tissue,  some  quite 
small  and  insular,  others  extending  over  a  considerable  area. 
These  patches,  which  are  characterised  by  an  accumulation  of 
lymphoid  cells  and  by  the  small  size  of  the  epithelial  cells  of 
the  surface,  are — at  least  the  larger  ones — provided  with 
numerous  blood-vessels,  the  epithelium  of  which  is  often  very 
well  stained  by  the  silver  ;  and  these  are  always  accompanied 
by  one  or  more  lymphatic  vessels,  with  walls  formed  by  the 
characteristic  wavy  outlined  cells. 

The  mesentery. — A  piece  of  the  mesentery  can  be  prepared 
in  a  manner  similar  to  that  employed  for  the  omentum.     But 


LY.M1']IATIC.S 


180 


the  glass  pl.ates  used  for  stretching  the  meiubiano  must  he 
smaller,  and  they  may  be  advantageously  replaced  by  Hoggan's 
rings.  These  consist  of  a  broad  and  slightly  conical  ring  of 
vulcanite,  an  inch  or  more  in  diameter,  over  the  smaller  end  of 
which  a  narrow  ring  slips.  This  smaller  end  is  applied  to  the 
piece  of  membrane  which  it  is  desired  to  use,  and  the  narrow 
ring  being  then  slipped  over,  the  membrane  is  securely  tixed 
and  stretched  over  the  larger  ring,  which  it  converts  into  a 
cup,  the  bottom  of  which  is  formed  by  the  membrane.  The 
remainder  of  the  tissue  is  cut  away.  Each  surface  of  the 
membrane  can  be  readily  brushed,  and  the  silver  nitrate  solu- 

FiG.  55 


tion  can  be  poured  into  the  cup,  and  thus  remain  in  complete 
contact  with  the  membi'ane.  A  wooden  ointment-box  with 
the  top  and  bottom  removed  forms  a  rough  substitute  for  the 
vulcanite  rings. 

The  central  tendon  of  the  diaphragm  with  its  serous 
coverings. — The  thoracic  and  abdominal  surfaces  of  the  dia- 
phragm present  important  difi'erences  in  the  arrangement  of 
the  numerous  lymphatic  vessels  which  are  distributed  upon 
them.  To  see  both  properly  it  will  be  necessary  to  sacrifice 
two  animals.  It  is  best  to  use  rabbits,  since  their  central 
tendon  is  larger  and  thinner  in  proportion  than  that  of  most 
other  mammals. 


190  PEACTICAL   HISTOLOGY 

For  the  preparation  of  either  side  both  thoracic  and 
abdominal  cavities  must  be  freely  opened,  the  marginal  attach- 
ment of  the  diaphragm  being  left  intact,  so  that  that  muscle 
remains  stretched  out.  A  double  ligature  is  to  be  put  on 
the  inferior  vena  cava  in  the  thorax,  and  the  vessel  cut 
between  the  two  threads  ;  this  is  to  prevent  the  blood  in  the 
vessel  from  getting  over  the  membrane.  The  pericardium  is 
now  cut  away  from  the  upper  surface  of  the  diaphragm,  and 
the  suspensory  ligament  of  the  liver  from  the  lower.  The 
side  upon  which  it  is  wished  to  display  the  lymphatics  is 
then  brushed  pretty  firmly  with  a  camel-hair  pencil  wetted 
with  nitrate  of  soda  solution  or  distilled  water,  after  which 
a  few  drops  of  nitrate  of  silver  solution  are  allowed  to  flow 
over  it,  or  are  applied  with  the  brush.  After  five  minutes' 
contact  the  silver  solution  is  washed  ofi"  by  a  stream  of  dis- 
tilled water,  and  the  central  tendon,  including  also  some  of 
the  muscular  fibres  which  converge  to  it,  is  carefully  removed, 
pinned  out  upon  a  loaded  cork  or  cake  of  wax,  with  the 
silvered  surface  uppermost,  and  exposed  to  the  sunlight  either 
in  water  or  50  per  cent,  spirit.  When  distinctly  browned 
it  is  removed  from  the  window,  and  pieces  from  different  parts 
are  cut  out  and  mounted  in  glycerine  or,  after  drying,  in 
xylol  balsam,  as  in  the  preparation  of  the  omentum.  The 
preparation  can  also  be  made  with  the  aid  of  Hoggan's 
rings. 

Stomata. — In  addition  to  these  preparations — which 
exhibit  the  lymphatics  and  cell-spaces  of  the  serous  mem- 
branes, and,  on  the  abdominal  side,  the  lymphatic  clefts 
between  the  tendon  bundles — it  is  useful  to  make  another 
silvered  preparation,  unbrushed,  of  the  peritoneal  surface, 
a  third  animal  being  sacrificed  for  the  purpose.  This  serves 
to  show  the  epithelial  layer  of  the  serous  membrane,  with 
the  differences  in  character  of  its  cells  in  diff'erent  parts,  the 
cells  being  much  smaller  over  the  interfascicular  lymphatics 
than  elsewhere.  Amongst  these  smaller  cells  may  be  seen 
here   and  there  the  minute   darkly-stained   angular  patches 


STOMATA  191 

known  as  pseudostonuita,  which  are  probably  merely  accumu- 
lations of  intercellular  substance ;  and  also — but  these 
are  more  dilHcult  to  find — the  true  holes  or  stomata  sur- 
rounded by  a  ring  of  small  cells,  and  leading  by  a  short  canal 
into  the  lymphatic  below.  Such  stomata  or  orifices  leading 
from  the  serous  cavities  into  lymphatic  vessels  are  met  with 
occasionally  in  preparations  from  most  of  the  serous  membranes 
in  mammals.  But  they  are  especially  numerous  and  well  seen 
in  the  peritoneum  of  the  frog. 

A  male  frog  should  be  killed  for  the  purpose,  and  the 
intestines  and  stomach  removed  so  as  to  expose  the  back 
of  the  abdomen,  but  without  cutting  the  mesentery  too  near 
the  spinal  column.  If  the  trunk  of  the  animal  is  now 
placed  in  a  dish  of  2  per  cent,  nitrate  of  soda  solution,  and  the 
posterior  pai't  of  the  peritoneum  carefully  examined  under 
that  fluid,  it  will  be  found  that  it  does  not  closely  cover  the 
vertebral  column,  great  vessels,  and  other  structures  which 
are  found  at  the  back  of  the  abdomen,  but  is  separated 
from  them  by  a  large  lyraph-space,  divided  from  the  serous 
cavity  by  a  membrane.  This  is  covered  on  the  one  side  by 
the  epithelial  cells  of  the  peritoneum  ;  on  the  other  by 
those  of  the  lymphatic,  and  to  the  unassisted  eye  appears 
to  form  a  complete  septum.  Under  the  microscope,  how- 
ever, it  is  seen  to  be  studded  by  very  numerous  apertures, 
which  can  be  seen,  even  in  the  fresh  condition,  if  the  mem- 
brane is  removed  and  examined  with  a  high  power.  But  to 
study  their  structure  and  the  arrangement  of  the  epithelial 
cells  with  reference  to  them,  and  also  to  obtain  a  permanent 
preparation,  the  septum  is  to  be  stained  with  silver.  With 
this  object  the  whole  septum,  or  a  portion  only,  is  dissected 
off  under  nitrate  of  soda  solution.  It  is  convenient  to  remove 
with  it  the  elongated  kidneys  which  adhere  to  it  behind,  and 
to  cut  these  away  only  after  the  staining  is  completed.  The 
membrane  is  placed  in  a  0*5  per  cent,  silver  nitrate  solu- 
tion for  one  minute,  again  rinsed,  and  exposed  in  water  to 
the  light.     After   the   metal   is   reduced  the   preparation    is 


192  PEACTICAL  HISTOLOGY 

floated  on  to  a  slide,  and,  with  the  usual  precautions  to  avoid 
folds  and  creases,  finally  either  mounted  in  glycerine  or  dried 
on  the  slide  and  mounted  in  xylol  balsam.  It  is  desirable, 
before  mounting,  to  stain  the  tissue  with  hsematein  so  as  to 
exhibit  the  nuclei  of  the  cells,  but  this  is  not  absolutely  neces- 
sary, and  much  increases  the  risk  of  producing  folds  in  the 
membrane. 

For  studying  the  structure  of  the  larger  lymphatic  vessels,  as, 
for  example,  the  thoracic  duct,  precisely  the  same  methods,  both 
for  teased  preparations  and  for  sections,  are  einployed  as  were  used 
for  the  larger  blood-vessels. 

INJECTION    OP    LYMPHATICS 

The  minute  lymphatics  of  a  part  may,  where  numerous, 
generally  be  readily  displayed  by  simply  sticking  a  very  fine 
cannula  into  the  tissue,  and  forcing  a  coloured  fluid  through 
this.  The  best  apparatus  for  the  purpose  of  obtaining  the 
requisite  pressure  is  the  small  mercury  apparatus  shown  in 
the  accompanying  figure  (fig.  56).  The  mercury  contained 
in  the  bottle  a  compresses  the  air  in  the  pressure-bottle  5, 
according  to  the  height  a  is  raised  above  &,  this  height 
being  regulated  with  the  greatest  nicety  by  the  screw  d. 
The  bottle  c  containing  the  injection-fluid  communicates  by 
one  tube  with  the  pressure-bottle,  and  by  another  (which 
passes  to  the  bottom)  with  the  injecting-cannula  f.  Gela- 
tine is  not  used  for  injecting  the  lymphatics,  but  almost 
always  injections  which  are  fluid  in  the  cold.  Berlin  blue 
solution  (2  per  cent.)  is  often  employed,  but  the  best  fluid 
for  the  purpose  is  a  solution  either  of  alkanet  or  of  asphalt  in 
turpentine,  either  of  which  readily  flows  into  the  lymphatics. 
The  cannula  can  be  made  from  a  piece  of  glass  tube  drawn 
out  to  a  capillary  point,  but  the  best  are  long  perforated  steel 
needles  like  those  supplied  with  hypodermic  syringes,  and 
as  fine  as  it  is  possible  to  procure  them  (fig.  57).  The  india- 
rubber  tube  connected  with  the  cannula  is  closed  by  the 
clip  g. 


INJECTION  OF  LYMPHATICS 


19:i 


The  mode  of  injecting  tlie  lymphatics  of  a  tendon  may 
be  here  described  as  an  example,  especially  as  the  subject 
was  deferred  when  studying  the  minute  structure  of  tendon. 


Fid.  r,v> 


Ludwig's  mercurial-pressure  apparatus  for  injecting  lymphatics 

rt,  reservoir  bottle  containing'  mercury  ;  6,  pressure-bottle  into  which  this  teutls  to  flow  : 
c,  iiijectiou-bottlc  containing-  solution  of  Berlin  blue,  connected  with  the  pressure- 
bottle  by  one  indiarubber  tube,  with  the  cannula  /"  by  a  second,  and  with  a  small 
manometer  by  a  third  ;  </,  handle  of  screw,  by  turning  which  the  sta<?e  on  which 
the  bottle  a  rests  is  raised  or  depressed,  iiiid  the  pressure  increased  or  diminished  in 
6  ;  e,  screw  clip  (opened) ;  </,  spring  cli])  (closed) 

One  of  the  best  tendinous  structures  to  choose  for  the 
purpose  is  the  fibrous  aponeurosis  covering  the  tendon  of 
the  quadriceps  extensor  femoris  of  the  dog.  Two  sets  of  lym- 
phatics are  here  met  with— one  in  the  substance  of  the  tendon, 

o 


194  PEACTICAL  HISTOLOGY 

consisting  for  the  most  part  of  vessels  arranged  conformably 
with  the  direction  of  the  fibres  and  connected  at  intervals 
by  transverse  branches,  so  as  to  form  elongated  and  oblong 
meshes  ;  and  a  superficial  one  in  the  areolar  sheath  which 
covers  the  aponeurosis,  consisting  of  vessels  forming  a  close 
plexus  with  polygonal  meshes.  The  latter  plexus  should  first 
be  attempted.  Both  tube  and  cannula  being  completely  filled 
with  the  injection-fluid  to  the  exclusion  of  air-bubbles,  the 
clip  g  is  closed,  and  the  cannula  is  inserted  obliquely  for 
half  an  inch  or  more  into  the  areolar  sheath,  care  being 
taken  not  to  discolour  the  surface  of  the  tissue  with  the 
injection-mass.  The  cannula  is  then  slightly  withdrawn  and 
the  clip  is  removed.  By  turning  the  handle  d,  and  thus 
raising  the  bottle  a,  the  pressure  is  put  on  to  about  an 
inch  of  mercury,  as  indicated  by  the  gauge  attached  to  the 

Fig.  57 


Very  fine  perforated  steel  needle  for  injecting  the  lymphatics  of  a  part 

injection-bottle.  If  the  insertion  of  the  cannula  have  been 
fortunate,  the  fluid  will  almost  immediately  begin  to  pass 
into  the  lymphatic  plexus,  but  should  there  be  no  result 
the  pressure  may  be  gradually  raised  to  about  two  inches  ; 
higher  than  this  it  is  not  as  a  rule  advantageous  to  go. 
If  there  is  still  no  result  the  cannula  may  be  pushed  a 
little  fui'ther  in  the  sheath,  and  perhaps  moved  a  little  to 
one  side  or  the  other  in  the  hope  of  thus  rupturing  a  lym- 
phatic and  gaining  an  entrance  into  the  plexus.  Should 
these  and  other  devices  which  experience  may  suggest  still 
fail,  the  clip  must  be  replaced  and  another  insertion  tried 
elsewhere.  It  very  frequently  happens  that  the  injection 
which  escapes  from  the  end  of  the  cannula,  instead  of  pass- 
ing into  the  lymphatics,  forms  merely  a  bulla  of  extrava- 
sated  flaid  in  the  interstices  of  the  tissue.     This  can  some- 


INJECTION   OF   LY3IPHATICS  195 

times,  by  passing  the  handle  of  a  scalpel  over  it  with 
moderately  firm  pressure,  be  induced  to  find  its  way  into 
the  absorbent  vessels,  but  if  not  the  cannula  must  be  with- 
drawn and  re-inserted  as  before. 

For  the  lymphatics  in  the  fibrous  substance  of  the  apo- 
neurosis the  cannula  must  be  inserted  obliquely  into  the 
tendinous  tissue,  and  the  injection  forced  in  with  the  same 
precautions.  The  pressure  may,  if  necessary,  be  raised 
somewhat  higher,  for,  owing  to  the  firmness  of  the  tissue, 
there  is  less  liability  to  extravasation. 

For  displaying  these  injected  preparations  they  may,  if 
injected  with  Berlin  blue,  be  first  placed  in  spirit  to  remove 
all  water  and  precipitate  the  colouring  matter  in  the 
vessels,  the  process  being  completed  by  putting  the  injected 
part  into  absolute  alcohol,  after  which  sections  may  be  cut, 
placed  ill  turpentine  and  mounted  in  xylol  balsam.  Another 
method,  and  one  which  succeeds  very  well,  especially  with 
the  alkanet  injection,  is  to  stretch  the  injected  aponeurosis 
over  a  ring  of  cork  and  allow  it  slowly  to  dry  by  exposure  to 
the  air.  When  completely  dry  the  injected  part  may  be  at 
once  mounted  in  glycerine.  By  this  mode  of  proceeding  the 
injection  is,  as  pointed  out  by  Bowditch,  rendered  more  com- 
plete, for  the  fluid  which  may  have  been  extravasated  in  the 
interstices  of  the  tissue  is  apt  to  become  drawn  into  the  lym- 
phatic vessels  to  supply  the  place  of  the  watery  fluid  which 
becomes  lost  by  evaporation. 

Preparations  injected  with  alkanet  or  asphalte  must  not 
be  put  in  turpentine,  but  always  mounted  in  glycerine. 

Although  in  many  cases  it  is  better  to  use  an  apparatus  of 
the  kind  above  described,  which  enables  the  pressure  which  is 
being  employed  to  be  exactly  estimated,  nevertheless,  with  a 
little  experience,  the  lymphatic  vessels,  especially  those  in  the 
firmer  tissues  and  organs,  may  often  be  injected  with  o-reat 
success  by  using  simply  an  ordinary  subcutaneous  syrinf^e 
provided  with  a  fine  cannula,  driving  the  injecting  fluid  into 
the  tissue  by  pressure  upon  the  piston.     It  is  true  that  extra- 

o2 


196  PEACTICAL  HISTOLOGY 

vasations  are  very  apt  to  be  produced  opposite  the  point  of 
the  cannula,  but  these  can  often  be  utilised  in  the  manner 
before  mentioned  by  gently  pressing  on  them  and  endeavouring 
to  induce  the  injection  to  pass  into  the  lymphatic  vessels. 

In  rare  cases  a  vein  is  pierced  by  the  cannula,  and  the 
system  of  blood  capillaries  of  the  part  is  then  apt  to  become 
filled,  but  both  the  vessels  themselves  and  the  meshes  they 
form  are  much  smaller  than  the  lymphatic  capillaries,  and 
a  knowledge  of  their  general  appearance  and  mode  of  arrange- 
ment in  the  particular  tissue  will  prevent  any  error  from 
arising  in  this  way. 

Injection  intra  vitam. — The  lymphatics  of  the  diaphragm 
may  be  injected  M'ith  Berlin  blue  during  life  : — A  young  rabbit 
is  chosen,  and  enough  chloral  hydrate  is  injected  under  its  skin 
to  anaesthetise  it  completely  (about  5  cubic  cent,  of  a  20  per 
cent,  solution  will  suffice).  The  skin  of  the  belly  is  then  cut 
through  for  a  couple  of  inches  close  to  the  ensiform  cartilage, 
the  edges  are  held  aside  by  an  assistant,  and  the  muscular 
wall  having  been  pinched  up,  a  cannula  is  passed  obliquely 
into  the  cavity  of  the  peritoneum  at  its  upper  part,  due 
care  being  taken  to  avoid  the  liver  and  stomach.  About 
five  cubic  centimetres  of  a  saturated  solution  of  Berlin  blue, 
previously  warmed,  is  now  injected  through  the  cannula, 
which  is  then  withdrawn  and  the  animal  put  aside  in  a 
warm  place.  After  four  hours,  during  the  whole  of  which 
time  it  remains  under  the  influence  of  the  chloral,  it  is  killed 
by  bleeding.  The  abdomen  is  then  opened,  and  the  viscera 
having  been  drawn  aside,  the  under  surface  of  the  diaphragm 
is  exposed,  and  the  blue  which  covers  it  is  washed  ofF  by  a 
stream  of  water.  If  the  experiment  has  been  successful, 
it  will  be  found  that  the  whole  network  of  lymphatics  of 
the  central  tendon  is  filled  with  the  blue  fluid ;  for  this, 
assisted  by  the  constant  respiratory  movements  of  the  dia- 
phragm, has  passed  from  the  peritoneal  cavity  directly  through 
the  open  stomata  into  the  lymphatic  vessels.  The  tendon  may 
be  cut  out  and  placed  in  alcohol,  and  eventually,  after  passing 


SYNOVIAL  ME.MBRANES  197 

tlii'Oiif^li  turpentiiio,  mounted  wliole  in  xylol  balsam,  Ijetwoen 
two  glass  plates,  and  used  for  examination  with  a  low  power 
of  the  microscope. 

THU    SYNOVIAL    MEMBRANES 

These  structures,  which  are  to  be  regarded  as  free  sur- 
faces of  the  ordinary  areolar  tissue  and  present  no  essential 
differences  in  structural  appearance  from  this,  may  be  prepared 
for  microscopical  examination  by  the  same  methods.  The 
preparations  which  are  of  greatest  value  are  those  stained 
with  nitrate  of  silver. 

Preparation  by  silver  method.— Since,  as  is  always  the 
case  with  the  silver  method,  the  parts  should  be  as  fresh  as 
possible,  and  since,  moreover,  it  is  convenient  to  have  large 
joints  to  work  with,  a  neat's  or  sheep's  foot  should  be  procured 
from  the  butcher's,  unless  a  freshly  amputated  human  limb  is 
available.  In  the  foot  all  three  kinds  of  synovial  membranes 
may  be  found  and  prepared.  The  mode  of  silvering  the  synovial 
bursa?  is  quite  simple,  and  need  not  here  be  detailed  ;  the 
preparation  of  the  vaginal  synovial  membranes  was  described 
under  Connective  Tissue  (p.  106),  this  being  taken  as  typical  of 
the  structure  of  that  tissue  ;  and  the  preparation  of  the 
synovial  surfaces  of  the  joints  was  given  under  Articular 
Cartilage  (p.  117).  But  in  the  last-mentioned  place  nothing 
was  said  as  to  the  mode  of  demonstrating  the  synovial  mem- 
brane proper,  for  we  had  there  to  do  only  with  the  cells  and 
cell-spaces  of  the  cartilage,  and  the  transitions  met  with 
between  these  and  the  cells  and  cell-spaces  of  the  synovial 
membrane.  The  appearance  presented  by  the  membi-ane 
itself  is  shown  in  surface  sections  made  from  the  inner  surface 
■of  the  capsule  of  the  joint.  It  will  be  seen  that  in  the  ox  the 
cells,  or  rather,  in  the  silvered  preparations,  the  white  cell- 
spaces,  form  a  close  irregular  network  by  the  union  of  their 
processes  ;  in  fact,  so  completely  have  the  cells  become 
extended  into  branches  that  without  staining  the  nuclei  it  is 
difficult  in  many  cases  to  make  out  the  situation  of  the  cell-body. 


198  PEACTICAL  HISTOLOGY 

In  the  human  synovial  membranes  this  is  not  the  case  ;  in  fact, 
the  appearances  are  quite  characteristic  of  ordinary  areolar 
tissue.  The  arrangement  of  the  corpuscles  into  epithelioid 
patches  is  not  infrequent,  but  there  is  no  continuous  epithelioid 
covering,  as  in  the  serous  membranes.  Moreover,  the  lym- 
phatics, which  are  so  numerous  in  serous  membranes,  are  not 
to  be  seen  in  the  synovial  membranes,  although  blood  capillaries 
are  present  and  in  many  places  approach  close  to  the  surface. 

Blood-vessels. — To  study  the  characteristic  arrangement 
of  the  blood-vessels  of  the  synovial  membranes  and  to  show 
the  circulus  artimili  vasctdosus,  a  preparation  must  be- 
made  from  one  of  the  joints  of  a  limb  that  has  been  fully 
injected.  Surface  sections  are  then  made  of  the  transitional 
region  where  the  synovial  membrane  terminates  on  the  carti- 
lage, and  including  also  a  part  of  the  membrane.  They  are 
mounted  in  the  usual  way  in  xylol  balsam,  without  being 
stained. 

Haversian  fringes. — Finally  the  Haversian  fringes,  with 
their  secondary  processes,  may  be  prepared.  They  may  be- 
examined  fresh  in  salt  solution,  and  may  also  be  obtained  from 
the  joint  which  was  stained  with  nitrate  of  silver.  To  find 
them  it  is  best  to  immerse  the  joint  in  fluid,  for  by  this 
means  they  are  floated  up,  and  may  then  be  snipped  off"  and. 
mounted. 


199 


CHAPTER  X 

THE    SKIX,    HAIRS,    AND   NAILS 

Sections  of  the  skin. — Portions  should  be  selected  for 
examination  from  dillerent  parts  of  the  body  ;  the  palms  of 
the  fingers  or  toes,  the  scalp,  and  a  piece  from  some  part  of 
the  general  surface,  e.g.  the  extensor  surface  of  the  forearm 
and  the  back  of  the  trunk.  The  skin  of  the  scrotum  may  also 
be  prepared,  to  show  the  bundles  of  plain  muscular  tissue  in 
the  subcutaneous  tissue  or  dartos,  and  a  small  piece  of  the  ala 
of  the  nose,  for  the  sake  of  the  well-marked  sebaceous  glands, 
which  open  into  the  follicles  of  the  minute  haii-s  found  in  this 
situation. 

The  following  method  of  hardening  the  tissue  may  be 
employed  : — A  small  piece  only  is  removed,  being  obtained 
Avith  as  little  of  the  subcutaneous  tissue  as  possible  adhering 
to  it.  At  the  same  time,  if  it  is  desired  to  examine  the  larger 
sweat-glands,  this  tissue  must  not  be  removed  too  freely, 
since  those  bodies  extend  down  into  it.  It  will  be  found  that 
the  fresh  skin  has  a  tendency  to  curl  in  at  the  edges ;  this 
should  be  prevented  by  pinning  the  piece  out  with  glass  pins 
or  hedgehog  spines  on  a  piece  of  cork.  The  latter  is  then  in- 
verted into  a  vessel  containing  saturated  solution  of  picric  acid. 
Corrosive  sublimate  and  75  per  cent,  spirit  also  answer  well. 
After  having  been  two  or  three  days  in  the  picric  acid  the 
piece  of  skin  is  transferred  to  spirit  containing  lithium  car- 
bonate, which  is  changed  until  tlie  excess  of  picric  acid  is 
extracted.     The  sections  may  be  cut  either  by  the  freezing  or 


200  PRACTICAL  HISTOLOaY 

the  paraffin  method,  preferably  the  former.  They  should  be 
vertical  to  the  surface,  but  a  piece  of  the  scalp  must  be 
embedded  so  as  also  to  be  cut  tangentially  to  the  surface. 
The  piece  from  the  palm  of  the  finger  is  to  be  cut  in  two 
directions,  viz.  (1)  across  and  (2)  parallel  with  the  ridges 
formed  by  the  papillse.  It  will  be  easier  to  cut  the  sections  if 
the  razor  is  made  to  travel  from  the  corium  towards  the 
epidermis  rather  than  in  the  opposite  direction.  The  pieces 
of  skin  which  contain  hairs  should  be  so  embedded  as  to  carry 
the  plane  of  the  vertical  section  in  the  direction  of  inclination 
of  the  hairs,  so  as  to  gain  a  view  of  the  hair  follicles  along 
their  whole  extent,  and  to  show  the  ao'recfores  pili  if  possible. 

The  sections  are  in  each  case  to  be  placed  in  carmalum 
or  logwood  solution,  and  when  sufficiently  stained,  to  be  washed 
in  water  and  transferred  successively  through  alcohol  and  oil 
of  cloves  to  balsam  in  the  usual  way.  Heidenhain's  method 
(p.  20)  also  gives  good  results. 

If  hardened  in  alcohol  or  sublimate  the  preparations  may 
be  much  improved,  so  far  as  the  exhibition  of  the  epidermis  is 
concerned,  by  being  placed  in  a  saturated  solution  of  picric 
acid  for  about  half  an  hour  and  then  thoroughly  washed  with 
distilled  water  before  being  stained,  or  in  alcohol  containing 
picric  acid  after  being  i^tained.  In  this  way  the  horny  parts 
acquire  a  bright  yellow  colour,  which  contrasts  strongly 
with  the  violet  staining  of  the  Malpighian  layer.  Owing  to 
the  number  of  nuclei  which  become  stained  in  it,  this  part  of  the 
epidermis  is  much  darker  than  the  papillary  part  of  the  corium 
which  is  in  contact  with  it.  In  the  papillae  of  the  skin  of  the 
finger  the  tactile  corpuscles  may  be  sought  for.  They  are 
generally  situated  quite  near  the  apex  of  a  papilla,  and  to  see 
them  well  it  is  important  to  cut  the  papillae  exactly  vertically,  so 
as  to  include  their  whole  length.  In  sections  which  have  been 
made  somewhat  obliquely,  and  in  sections  cut  parallel  instead 
of  vertical  to  the  surface,  the  transversely  or  obliquely  cut 
papillae  appear  as  round  or  oval  islands  in  the  midst  of  the 
deeper  cells  of  the  epidermis.     The  denticulated  appearance 


TilE   SKIN  201 

presented  by  most  of  the  colls  of  the  jNIalpighiaii  layei-,  and 
the  intercellular  channels  can  i-eadily  be  seen  with  a  higli 
power  immersion  objective. 

Of  the  sections  made  from  the  skin  of  the  linger  one  «r 
two  of  the  thinnest,  after  being  stained  with  picrocarmine,  may 
be  mounted  in  glycerine.  The  fibrous -Idoking  tactile  corpuscles 
can  generally  be  made  out  better  in  these  than  in  the  prepara- 
tions which  have  been  mounted  in  balsam. 

Blood-vessels. — To  sliow  the  arrangement  of  the  blood- 
vessels, sections  of  skin  from  a  limb  which  has  been  minutely 
injected  may  be  jnade  and  mounted  in  balsam  by  the  usual 
process.  These  must  either  be  left  entirely  unstained  or  the 
staining  must  be  slight.  The  sections  will  generally  include 
clusters  of  fat-cells,  with  their  vessels. 

Hairs. — To  examine  a  hair,  all  that  is  necessary  is  tu  place 
it  on  a  slide  in  a  drop  of  Avater,  cover  with  a  thin  glass,  and 
examine  with  a  mcjderately  high  magnifying  power.  By 
careful  focussing  the  cuticular  scales  can  be  made  out  on 
the  surface  and  at  the  edges  of  the  hair,  especially  on  the 
small  hairs  of  the  general  surface  of  the  body.  The  medulla 
is  often  absent  in  hairs  of  the  head,  but  may  generally  be 
found  in  those  of  the  beard  and  whiskers.  Many  of  the 
black  particles  which  are  seen  in  a  hair  by  reflected  light, 
especially  in  the  medulla,  are  merely  small  globules  of  air  in 
the  interstices  of  the  tissue.  That  this  is  so  may  be  proved 
by  cutting  off  the  light  which  comes  from  the  mirror  of  the 
microscope  and  viewing  the  object  by  reflected  light,  only  a 
moderate  power  being  used.  Tlie  l)lack  particles,  if  really  due 
to  the  presence  of  air,  will  then  appear  silvery  white,  just  as 
in  the  pai'allel  case  of  the  air  which  Alls  the  lacuiue  in  a 
section  of  hard  bone. 

It  will  be  useful  to  compare  the  appearances  presented  by 
iiuman  hair  with  those  exhibited  by  the  hairs  of  some  of  the 
common  domestic  animals.  These  are  many  of  th(>m  charac- 
terised by  the  regular  arrangement  of  the  medulla  (this  is 
nearly  always  present  in  the  hairs  of  quadrupeds),  which  forms 


202  PEACTICAL  HISTOLOGY 

different  patterns  in  different  kinds  of  animals,  so  that  the 
species  to  which  the  hair  belongs  may  often  be  determined. 

The  fibrous  part  of  a  hair  can  be  broken  up  into  its  con- 
stituent fibres  and  cells  if  it  be  first  steeped  for  a  time  in 
strong  sulphuric  acid. 

The  relative  proportion  of  the  three  constituent  parts 
of  a  hair  to  one  another  is  best  shown  in  transverse  sections. 

To  obtain  sections  of  hairs,  the  simplest  plan  is  to  tie  a 
number  together  and  dip  the  bunch  into  strong  clear  gum,  and 
when  this  has  thoroughly  soaked  in  amongst  the  hairs  to 
remove  the  bunch,  and  either  let  the  gum  dry  and  harden  by 
exposure  to  the  air  or  plunge  it  into  spirit  containing  one- 
sixth  water,  by  which  in  a  few  hours  the  whole  mass  is 
rendered  hard.  Sections  are  then  made  with  a  microtome  pro- 
vided with  a  sharp  plane-iron,  and  are  mounted  in  glycerine. 

Hair-follicles  and  roots  of  hairs  are  seen  in  the  sections 
of  skin,  especially  those  of  the  scalp. 

Nails. — The  nails  are  studied  by  means  of  vertical  sections 
made  both  longitudinally  and  transversely.  The  finger  (or 
toe)  should  if  possible  be  previously  injected,  and  the  nail 
with  the  matrix  and  surrounding  skin  having  been  removed, 
may  be  hardened  either  in  spirit  or  sublimate  or  by  picric 
acid  and  cut  frozen  after  being  soaked  with  gum.  When 
ready  for  cutting,  the  piece  should  be  bisected  longitudinally  ; 
and  from  one  of  the  halves  longitudinal  sections  (which  need 
not  be  very  thin)  are  taken  along  the  whole  length,  to  show 
the  general  relation  of  the  nail  to  its  matrix  and  to  the  epi- 
dermis. These  may  be  stained  or  left  unstained ;  they  may 
be  mounted  either  in  glycerine  or,  if  injected,  in  balsam  ;  they 
are  intended  chiefly  for  examination  with  a  low  power.  The 
other  piece  is  to  be  placed  on  the  microtome  in  such  a  way 
that  the  laminae,  which  in  the  matrix  represent  the  papillae  of 
the  skin,  are  cut  transversely.  The  sections  must  be  as  thin 
as  possible,  and  stained  either  with  picrocarmine  or  with  log- 
wood, or  with  carmalum  followed  by  alcoholic  solution  of 
picric  acid. 


TUE   SKIN  203 

But,  owing  to  the  substance  of  the  nail  being  so  much 
harder  than  the  subjacent  matrix,  it  is  very  difficult  to  get 
both  parts  equally  thin.  They  can,  however,  be  g(jt  of  much 
the  same  degree  of  hardness  by  means  of  the  gum-alc(jhol 
method.  The  piece  to  be  cut,  which  should  be  quite  small, 
is  placed  in  syrupy  solution  of  gum  and  left  for  two  or  three 
days  ;  it  is  then  transferred  to  a  mi.xture  of  spirit  with  one- 
sixth  of  its  volume  of  water.  After  a  few  hours  the  gum, 
which  has  penetrated  into  the  substance  of  the  tissue,  will  be 
hardened  throughout,  and  the  mass  can  be  fixed  in  a  micro- 
tome and  cut  in  the  desired  direction,  the  plane  which  is  used 
being  wetted  with  some  of  the  same  spirit-mixture.  Strong 
spirit  should  not  be  used,  since  the  gum  is  entirely  dehydrated 
by  this,  and  becomes  so  hard  as  to  turn  the  edge  of  the  knife. 
The  sections  are  transferred  from  the  spirit  to  water,  which 
dissolves  out  the  gum  ;  when  quite  free  from  this  they  are 
stained  and  mounted  as  before. 

Nerves  to  the  skin  and  its  appendages.— The  nerve 
terminations  in  the  epidermis,  in  the  hair-follicles,  in  the 
sweat-glands,  and  in  other  parts  of  the  skin,  may  be  studied 
either  by  Lowit's  gold  chloride  method  (p.  159)  or  by  Golgi's 
silver  chromate  method  (p.  152),  or  by  Ehrlich's  methylene 
blue  method  (p.  160). 

Lymphatics. — The  lymphatics  of  the  skin  may  be  studied 
either  in  preparations  made  by  the  method  of  interstitial  in- 
jection (p.  192)  or  in  silvered  preparations.  The  latter  can 
be  best  obtained  from  the  skin  of  a  small  animal,  such  as  the 
mouse.  A  piece  of  the  skin  is  stretched  over  a  Hoggan's  ring 
(fig.  55),  and,  the  hair  having  been  shaved  off,  it  is  immersed 
in  1  per  cent,  nitrate  of  silver  solution  for  15  minutes  ;  then 
washed  with  distilled  water  and  exposed  to  the  light.  When 
brown  it  is  placed  in  spirit  to  dehydrate  it,  then  in  clove 
oil,  and  finally  a  piece  is  cut  out  and  mounted  whole  in  xylol 
balsam. 

Development  of  hairs  and  skin-glands.— To  study  the  de- 
velopment of  hairs  and  of  the  skin  generally,  sections  of  parts 


204  PEACTICAL  HISTOLOGY 

of  foetal  animals,  •which  have  been  hardened  in  0'2  per  cent. 
chromic  acid  or  picric  acid  (saturated),  may  be  employed. 
Many  stages  in  the  development  of  hairs  are  seen  in  such 
sections  from  the  new-born  rat.  The  tissue  should  be  stained 
in  bulk  and  embedded  in  the  usual  manner  in  paraffin.  The 
sections  must  be  fixed  to  the  slide  (p.  37),  and  if  found  to 
be  over-stained  the  excess  of  stain  can  then  be  removed  by 
the  cautious  use  of  acid  alcohol. 


205 


CHAPTER  XI 

THE    HEART 

The  cardiac  pericardium. — The  pericardium  which  covers 
the  surface  of  the  heart  is  prepared  by  the  same  methods  as 
other  serous  membranes.  Of  these  the  only  one  which  need 
here  be  described  is  that  by  nitrate  of  silver.  This  is  as 
follows  : — In  an  animal  which  has  just  been  killed  the  thorax 
is  freely  opened,  and  the  pericardium  having  been  cut  open, 
the  base  of  the  heart  is  secured  by  a  tape  ligature,  the  great 
vessels  being  then  cut  beyond  the  ligature,  and  the  organ  re- 
moved without  allowing  its  surface  to  be  smeared  with  blood. 
A  part  of  the  surface  is  now  brushed  firmly  with  a  soft  camel- 
hair  brush  moistened  with  2  per  cent,  nitrate  of  soda  solution, 
witli  the  object  of  removing  the  superficial  layer  of  epithelial 
cells.  The  heart  is  next  rinsed  with  the  same,  and  nitrate  of 
silver  solution  is  poured  over  the  whole  surface,  and  allowed 
to  be  on  it  for  three  minutes,  after  which  the  organ  is  rinsed 
in  distilled  water,  and  finally  placed  in  spirit,  in  the  sunlight. 
When  sufficiently  browned  it  is  removed  from  the  window, 
and  left  for  some  hours  until  the  surface  is  hardened  by  the 
alcohol.  Surface  sections  are  then  made  of  both  unbrushed 
and  brushed  parts,  and  after  being  passed  through  clove  oil 
are  mounted  separately  in  balsam.  The  unbrushed  specimens 
will  show  the  epithelial  layer  ;  the  brushed  ones  should 
exhibit  the  subjacent  connective  tissue,  with  its  cell-spaces, 
lymphatics,  blood-vessels,  and  nerves. 

The  muscular  substance  of  the  heart  is  studied  in  teased 
preparations  and  in  sections.     For  the  teased  preparations  the 


206  PEACTICAL  HISTOLOGY 

heart  of  a  young  animal  should  be  chosen,  since  in  these  the 
fibres  separate  more  readily  into  their  constituent  cells,  A 
small  shred  is  placed  in  a  solution  of  picrocarmine  for  ten  days 
or  more  ;  it  is  then  broken  up  in  water  as  minutely  as 
possible,  and  glycerine  added  and  the  preparation  covered. 
Numerous  little  fragments  of  varying  shapes  will  be  found 
scattered  over  the  preparation.  On  careful  examination  it 
will  be  apparent  that  each  possesses  a  nucleus.  These  little 
fragments  of  the  cardiac  muscular  tissue,  which  have  the 
characteristic  indistinct  striation  of  that  substance,  are  the 
cells  which  by  their  union  end  to  end  form  the  fibres. 

To  show  the  arrangement  of  the  fibres,  and  the  interstitial 
tissue  and  vessels,  a  piece  of  the  muscular  substance  is  to  be 
placed  in  strong  spirit.  In  two  or  three  days  it  will  be 
ready  to  embed  and  cut.  Sections  are  to  be  made  both  parallel 
with  and  across  the  direction  of  the  fibres  ;  they  are  to  be 
stained  with  hsematein  and  mounted  in  balsam. 

The  endocardium. — To  display  the  endocardium  the  silver 
method  again  comes  into  requisition.  The  part  of  the  lining 
membrane  which  covers  the  septal  wall  of  the  right  ventricle  is 
the  best  to  prepare,  on  account  of  its  relative  smoothness. 
The  right  ventricle  is  opened  in  a  fresh  heart,  and  the  outer 
wall  removed  entirely,  and  then  a  large  piece  of  the  smoothest 
part  of  the  exposed  surface  of  the  septum  is  sliced  off  with  a 
razor.  A  part  only  of  the  endocardium  of  the  detached  piece 
is  brushed,  as  in  the  case  of  the  pericardium,  and  the  whole  is 
then  washed  and  treated  with  silver  solution.  After  three 
minutes  it  is  put  into  spirit  as  before,  and  when  browned 
and  hardened,  surface  sections  are  cut  and  mounted  in  balsam. 

In  addition  to  these  silvered  preparations,  the  endocardium 
should  be  examined  in  the  fresh  state.  This  is  done  by  dis- 
secting ofi"  a  piece  of  the  membrane  in  salt  solution  and  ex- 
amining it  both  with  and  without  the  addition  of  acetic  acid. 
Other  portions  may  be  teased  out  with  a  view  to  the  demon- 
stration of  the  elastic  and  muscular  tissue.  The  methods  for 
making  and  preserving  these  preparations  are  the  same  as 


THE  HEART  207 

were  employed  foi-  sliowing  the  structure  of  tlie  coats  of  the 
blood-vessels,  to  the  description  of  which  the  student  is  referred 
(p.  164).  It  may  be  noted  that  in  some  animals — the  sheep, 
for  instance— the  peculiar  large  cubical  or  oblong  cells  wliicli 
form,  in  series,  the  fibres  of  Purkinje  will  be  found  in  the 
endocardium.  They  are  about  the  size  of  fat-vesicles,  which 
are  also  found  in  the  endocardium  in  this  animal ;  but  the  two 
can  hardly  be  confounded,  for  the  cells  forming  Purkinje's 
fibi-es  have  a  clear  or  slightly  granular  binucleated  central 
portion  which  does  not  strongly  refract  the  light,  and  a  striated 
circumference,  which  is  appaiently  continued  into  that  of  the 
neighbouring  cells  ;  whereas  the  fat-cells,  although  they  may 
also  occur  in  rows,  and  may  be  of  much  the  same  size  as  the 
cells  in  question,  present,  by  virtue  of  their  strong  refracting 
power  on  light,  a  totally  different  appearance. 

Lymphatic  system  of  the  heart— If  the  tine  cannula  of  a 
hypodermic  syringe  tilled  with  Berlin  blue  solution  is  stuck  into 
the  muscular  substance  of  the  fresh  heart  at  any  part,  and 
the  fluid  is  forced  out  at  the  point,  the  injection  will  pass 
freely  into  the  lymphatic  interstices  between  the  muscular 
fibres,  and  if  the  tube  is  inserted  near  the  outer  or  inner  sur- 
face, will  find  its  way  into  the  lymphatics  of  the  pericardium 
or  endocardium,  which  can  in  this  way  be  readily  displayed. 

Blood-vessels. — The  blood-vessels  of  the  heart  are  to  be 
studied  in  sections  from  an  injected  preparation.  The  nerves 
may  be  stained  by  the  silver  chromate,  or  by  the  methylene 
blue  method,  but  they  are  somewhat  difficult  to  demonstrate 
satisfactorily. 


208  PEACTICAL  HISTOLOGr 


CHAPTER  XII 

THE   LUNGS 

The  pulmonary  pleura. — The  serous  membrane  which 
covers  the  surface  of  the  lungs  as  well  as  that  which  lines  the 
wall  of  the  thorax  is  prepared  by  the  silver  process.  For  the 
pulmonary  pleura,  the  lungs  of  a  small  animal  that  has  just 
been  killed  are  to  be  removed  entire,  and  moderately  distended, 
with  air  through  the  wind-pipe,  the  bronchi  being  then  tied 
and  the  two  lungs  separated.  One  is  rinsed  for  a  moment  in 
distilled  water,  and  a  little  nitrate  of  silver  solution  is  allowed 
to  flow  over  the  surface  ;  after  the  lapse  of  a  minute  this  is 
washed  off  again  with  distilled  water,  and  the  organ  is  then 
immersed  in  a  beaker  of  spirit  and  exposed  to  the  light. 

The  surface  of  the  other  lung  is  to  be  firmly  brushed  with 
a  wet  camel-hair  pencil,  to  remove  the  epithelial  cells  of  the 
surface  before  treating  it  with  the  silver  solution.  This  may 
be  suffered  to  remain  longer  in  contact  with  it  than  with 
the  other  lung  (5  minutes) ;  in  other  respects  the  treat- 
ment is  similar.  Both  preparations  are  left  in  the  light 
until  they  appear  sufiiciently  stained,  after  which  they  are  to 
be  placed  on  one  side  in  the  spirit  for  twenty-four  hours. 
They  will  then  be  sufficiently  hard  to  render  it  possible  to 
shave  off  a  thin  slice  from  the  surface.  The  sections  so  made 
are  to  be  mounted  in  balsam,  with  the  outer  surface  upper- 
most. 

The  costal  pleura  is  to  be  prepared  in  situ  after  the  re- 
moval of  the  lungs  and  heart.  That  of  one  side  may  be 
brushed,  the  other  not  ;  on  the  latter  the  silver  solution  is,  as 


THE   LUNGS  209 

before,  to  be  allowed  to  remain  a  shorter  time  than  on  thcj 
brushed  part,  where  the  fluid  has  to  penetrate  into  the  lym- 
phatic vessels,  and  into  the  substance  of  the  tissue.  The 
whole  thorax,  if  small  {ejj.  mouse),  )nay  then  be  cut  ofi'  from  the 
rest  of  the  trunk  and  exposed  in  spirit  to  the  light  ;  or,  if  too 
large  to  do  this  conveniently,  a  piece  only  of  the  thoracic 
parietes  on  each  side  is  to  be  removed  and  pinned  out  on  to  a 
cork,  which  is  then  placed  in  a  vessel  of  spirit  in  the  sunlight. 
When  stained,  pieces  of  the  membrane  must  be  carefully  dis- 
sected off,  without  pulling  upon  or  injuring  the  tissue  in  any 
way,  floated  upon  a  slide,  the  excess  of  spirit  poured  off,  all 
creases  removed  from  the  membrane,  which  is  then  allowed  to 
dry,  and  finally  mounted  in  balsam. 

The  lung  tissue. — Tlie  structure  of  the  lungs  themselves  is 
best  shown  by  means  of  sections.  The  tissue  is  hardened  in 
the  following  way  : — 

The  organs  having  been  removed  from  the  chest  of  a 
recently  killed  animal,  care  being  taken  not  to  scratch  their 
surface  with  the  broken  ends  of  the  libs,  a  glass  cannula  is 
tied  into  the  end  of  the  trachea  (or  into  either  bronchus). 
The  cannula  is  then  connected  by  an  indiarubber  tube  with  an 
injection-bottle,  which  is  filled  with  a  weak  solution  of  chromic 
acid  (0.2  per  cent.)  ;  this  is  made  to  flow  into  the  lungs  so 
as  to  distend  them  moderately.  The  trachea  or  bronchus  is 
now  tied  up,  the  cannula  removed,  and  the  lungs  are  immersed 
in  a  large  quantity  of  a  solution  of  chromic  acid  of  similar 
strength.  After  a  few  hours  the  fluid  is  changed,  and  the 
organs  are  cut  into  pieces,  to  enable  the  fresh  fluid  moi-e 
readily  to  penetrate.  After  a  day  or  two  more  in  this  the 
pieces  are  placed  first  for  twenty-four  hours  in  weak  spirit, 
and  then  in  strong  spirit,  which  may  again  be  changed  once 
or  twice.  Picric  acid  or  corrosive  sublimate  (saturated),  formol 
(o  per  cent.),  or  bichromate  of  potash  (3  per  cent.),  may  be 
used  in  place  of  chromic  acid. 

The  pieces  should  be  stained  in  bulk  with  dilute  hanna- 
tein    or   with    carmalum,  or  by    Heidenhain's    method,    and 

p 


210  PEACTICAL  HISTOLOGY 

embedded  in  paraffin.  Sections  may  be  made  both  across 
and  along  the  course  of  the  main  bronchial  tubes,  and,  after 
going  through  the  usual  processes,  mounted  in  balsam. 

These  stained  sections  of  lung  may  be  first  examined 
with  a  moderate  power,  but  afterwards  a  power  of  400  or  500 
diameters  should  be  employed,  in  order  to  see  the  details  of 
structure  ;  the  ciliated  epithelium,  muscular  layer,  and  carti- 
laginous plates  of  the  bronchial  tubes,  with  the  mucous  glands, 
nerves,  lymphatics  (seen  in  section  as  mere  clefts),  and  patches 
of  lymphoid  tissue  in  their  walls  ;  the  branches  of  the  pulmonary 
artery  accompanying  them  ;  the  mode  in  which  the  terminal 
air-tubes  dilate  into  the  infundibula  ;  the  air-cells  or  alveoli, 
almost  covered  with  a  network  of  capillaries,  which  are  seen 
also  on  the  septa  between  the  alveoli,  projecting  first  into  one 
and  then  into  the  other  of  two  neighbouring  air-cells.  Where 
they  run  vertically  the  capillaries  appear  in  optical  section  as 
circular  spots,  looking  not  unlike  nucleated  cells.  But  the 
excessively  delicate  epithelium  of  the  air-cells  cannot  be  well 
seen  in  these  preparations,  for  the  epithelium  cells  remain 
almost  unstained,  and  it  is  not  easy  to  differentiate  their 
nuclei  from  those  of  the  closely  subjacent  capillaries. 

Epithelium  lining  the  air-cells. — In  order  to  demonstrate 
the  epithelium  cells  nitrate  of  silver  is  made  use  of,  but 
the  mode  of  proceeding  is  somewhat  different  from  that 
ordinarily  employed.  A  gelatine  mixture  is  made  by  taking 
ten  grammes  of  gelatine,  and,  after  soaking  it  in  cold  distilled 
water,  melting  it,  and  adding  if  necessary  more  warm  distilled 
water  until  the  mixture  measures  100  c.c.  A  decigramme  of 
nitrate  of  silver  is  dissolved  in  a  little  distilled  water  and 
added  to  the  gelatine,  and  the  mixture  is  transferred  to  an 
injection-bottle,  which  is  kept  warm  over  a  water-bath. 
The  injection-bottle  is  provided  with  an  indiarubber  cork 
and  two  glass  tubes,  one  of  which  serves  to  blow  air  into  the 
bottle  and  thus  raise  the  pressure,  and  the  other  to  conduct 
the  gelatine  by  means  of  an  indiarubber  tube  to  a  cannula  tied 
into  the  trachea  or  into  a  bronchus.     An  animal — preferably 


THE   LUNGS  211 

;i  younf,'  one — having  been  killed  l)y  bleeding,  the  lungs  ;ii-e 
removed,  the  glass  cannula  is  tied  into  the  trachea,  and 
enough  of  the  gelatine  mixture  is  injected  into  the  lungs  to 
distend  tlicm  pretty  completely.  The  trachea  is  now  tied  and 
the  cannula  removed  from  it.  The  lungs  are  then  put  aside 
into  a  cold  place  until  the  gelatine  within  them  has  fully 
set,  when  sections,  which  should  be  as  thin  as  possible,  are 
made  with  a  razor,  either  not  wetted  at  all  or  with  distilled 
water  only.  The  sections  so  obtained  are  placed  on  a  slide  in 
glycerine,  covered,  and  exposed  to  the  light.  As  soon  as  they 
seem  sufliciently  stained  they  may  be  examined  with  as  high  a 
power  as  possible,  for  the  purpose  of  making  out  the  silver - 
lines  between  the  epithelium  cells. 

Blood-vessels  of  the  lungs. — The  pulmonary  vessels  are  to 
be  injected  and  sections  made  of  the  injected  lung.  The  red 
gelatine  injection  may  be  used  ;  this  and  everything  else  is  to 
be  got  ready  in  the  same  way  as  for  the  injection  of  the  aortic 
system,  but  the  trachea  must  be  clamped  in  the  neck  before 
the  chest  is  opened,  in  order  to  prevent  the  lungs  from 
collapsing.  The  arterial  cannula  is  of  course  to  be  passed 
through  the  rigid  ventricle  and  tied  into  the  pulmonary  artery 
instead  of  into  the  aorta.  The  gelatine  injection  is  now  set 
flowing  through  the  pulmonary  vessels,  the  lungs  being  still 
distended  with  air  ;  the  pressure  is  then  raised  in  the  injecting- 
apparatus  to  about  three  inches  of  mercury.  The  left  ventricle 
is  first  slit,  to  let  the  blood  out  of  the  pulmonary  system,  and 
then  clamped,  to  prevent  the  escape  of  the  injecting-fluid,  which 
is  allowed  to  pass  until  it  is  thought  that  the  vessels  must  all  ])e 
completely  filled.  The  trachea  is  now  opened  so  that  the  lungs 
collapse,  but  they  are  brought  back  to  their  previous  condition 
by  the  injection  of  warm  3  p.c.  potassium  bichromate  solution 
into  them  through  the  windpipe.  The  trachea  and  the  base 
of  the  heart  are  then  ligatured,  and  the  whole  is  left  to  cool. 
The  lungs  are  then  cut  out  and  placed  in  3  p.c.  potassium 
bichromate  ;  after  a  few  days  sections  (not  too  thin)  may  be 
made   by  the  freezing  method  and  mounted  in    balsam.     If 

p2 


212  PEACTICAL  HISTOLOGY 

it  is  desired  to  stain  the  tissue  somewhat,  so  as  to  show 
the  general  structure  of  the  lung  as  well  as  the  arrangement  of 
the  blood-vessels  in  the  same  preparation,  this  can  be  done  by 
hsematein. 

The  injected  lung  may  also  be  distended,  while  the  injection 
is  still  flowing,  with  melted  paraffin,  and  when  cold  placed  in 
alcohol,  the  sections  being  cut  by  the  paraffin  method. 

Larynx  and  trachea. — The  trachea  and  larynx  are  hardened 
in  0"2  per  cent,  chromic  acid,  the  hardening  being  completed 
by  spirit,  as  with  the  lung.  If  not  previously  stained  in 
bulk  the  sections,  which  may  be  longitudinal  of  the  carti- 
laginous part  and  transverse  of  the  posterior  membranous  part, 
are  to  be  stained  with  logwood  or  carmalum  and  mounted  in 
balsam. 

Blood-vessels,  lymphatics,  and  nerves  of  trachea.  — In 
addition  to  these  sections  flat  preparations  showing  the  blood- 
vessels, and  others  showing  the  lymphatics  of  the  mucous 
membrane,  may  be  made.  The  former  are  got  from  any  animal 
that  has  been  injected  entire,  the  mucous  membrane  being- 
dissected  off"  and  mounted  in  balsam.  The  lymphatics  are 
readily  filled  by  the  puncture  method  by  sticking  the  point  of 
the  injecting  cannula  into  the  mucous  membrane,  and  forcing 
in  a  little  Berlin  blue  or  alkanet-turpentine.  It  will  hardly 
ever  fail  in  finding  its  way  into  the  numerous  lymphatics  of 
the  mucous  membrane.  The  injected  portion  is  dissected  off" 
and  mounted  in  balsam  or  in  glycerine.  The  meduUated 
nerves  may  be  shown  by  merely  exposing  the  fresh  mucous 
membrane  to  vapour  of  osmic  acid  (Stirling)  ;  their  fine  ter- 
minations by  the  silver  chromate  or  methylene  blue  method. 

Teased  preparations  to  show  the  separated  epithelial  cells 
have  already  been  described  (p.  94). 


213 


CHAPTER   XIII 

Till':  MOUTH  AND  PHARYNX 

Mucous  membrane  of  the  mouth. — Portions  of  the  lining 
membrane  of  the  mouth  are  best  prepared  by  being  pinned  out 
upon  a  cork  and  placed  in  strong  spirit  or  absolute  alcohol. 
Sections  of  the  cheek  or  lip  may  also  thus  be  readily  pre- 
pared. 

THE    TEETH 

Sections  of  hard  tooth. — No  preparations  exhibit  the 
general  structure  of  the  teeth  better  than  these.  A  slice  cut 
with  a  fine  saw  from  the  hard  tooth  is  ground  down  first  on 
one  side  and  then  on  the  other,  until  a  thin  section  only  re- 
mains, and  this  is  mounted  in  hard  Canada  balsam  in  such  a 
way  that  the  air  still  remains  in  the  dentinal  tubules,  the  lacunar 
of  the  cement,  the  interglobular  spaces,  and  other  minute  cavi- 
ties that  may  be  present.  The  preparation  is  similar  to  that 
of  bone,  but  presents  greater  difficulty.  Such  specimens  may 
advantageously  be  purchased,  for  their  jDreparation  involves  the 
expenditure  of  a  large  amount  of  time  and  labour  ;  unless  the 
use  of  a  lapidary's  wheel  can  be  obtained,  when  the  process 
is  much  facilitated.  They  should  in  every  case  be  studied 
first  with  a  low  power,  and  afterwards  with  a  liigh  power 
objective. 

Von  Koch's  method. — Another  method  of  preparing  sections  of 
liard  teeth  with  the  soft  parts  preserved  in  situ  is  that  devised 
by  V.  Koch  for  obtaining  thin  sections  of  calcareous  sponges  and 
corals,  and  applied  to  the  histologj^  of  the  teeth  by  Weil.     The 


214  PEACTICAL  HISTOLOGY 

tooth  is  preserved  by  being  placed  in  absolute  alcohol  for  a  few 
days  ;  other  fixing  fluids  may  be  used :  e.g.  Hermann's  or  Plem- 
ming's,  but  they  should  be  followed  by  alcohol.  It  is  stained  in 
bulk  by  being  placed  for  several  days  in  picrocarmine,  or  in 
carmalum.  It  is  then  dehydrated  by  alcohol  of  gradually  in- 
creasing strength,  finishing  up  with  absolute  alcohol,  and  being 
subsequently  placed  in  chloroform.  From  this  it  is  transferred  to  a 
strong  solution  of  hard  Canada  balsam  in  chloroform,  in  which  it 
also  remains  for  several  days.  When  removed  it  is  allowed  to  dry 
slowly,  until  the  Canada  balsam  has  become  hard  throughoiit. 
Sections  are  now  made  with  a  fine  saw,  and  are  ground  down  with 
the  aid  of  a  lapidary's  wheel,  until  they  appear  thin  enough  ;  they 
are  then  moimted  in  Canada  balsam. 

Sections  of  softened  tooth. — But,  in  addition  to  the  facts 
which  the  hard  specimens  will  show,  various  others  may  be 
made  out  in  teeth  which  have  been  softened  by  immersion  in 
an  acid.  The  methods  of  decalcification  are  the  same  as  for 
bone  (pp.  125  to  127). 

The  acid  generally  used  when  the  structure  of  the  (decal- 
cified) dentinal  substance  only  is  to  be  investigated  is  nitric  or 
hydrochloric.  A  nearly  saturated  solution  of  common  salt 
containing  10  per  cent,  of  either  of  these  acids  may  be  em- 
ployed, and  the  tooth  is  steeped  in  this  until  entirely  soft,  after 
which  it  is  placed  in  the  salt  solution  without  acid.  After 
several  changes  into  fresh  quantities  of  this,  it  may  be  preserved 
in  spirit.  Sections  are  to  be  made  in  planes  both  parallel  with 
and  across  the  direction  of  the  dentinal  tubules. 

Dentinal  sheaths. — To  show  the  sheaths  which  line  the 
dentinal  tubules  a  piece  of  such  softened  tooth  is  transferred 
to  strong  hydrochloric  acid  (contained  in  a  watch-glass,  which 
is  covered  by  another,  inverted)  and  left  for  about  an  hour, 
after  which  time  all  that  will  be  found  is  a  tenacious  soft  mass 
occupying  its  place.  If  some  of  this  mass  be  removed  with  a 
small  pointed  piece  of  wood,  placed  on  a  slide,  covered,  and 
examined,  it  will  be  found  to  consist  wholly  of  fine  tubular 
threads — the  dentinal  sheaths.  These  being  composed  of  a 
substance  which  resists  the  solvent  action  of  strong  hydro- 


THE  TEETH  215 

chloric  acid  longer  than  the  other  animal  tissues,  remain  for  a 
time  visible  after  the  rest  of  the  dentinal  substance  has  dis- 
appenved. 

Soft  tissues  of  the  teeth.  -  But  to  study  the  soft  tissues — 
that  is  to  say,  the  pulp  and  odontoblasts  with  the  processes 
which  these  send  into  the  dentinal  tubules — we  must,  as  in 
the  parallel  case  of  bone,  employ  a  reagent  which,  whilst 
softening  the  hard  parts,  at  the  same  time  preserves  and 
hardens  the  soft  parts.  Phloroglucin-nitric  acid  (p.  127)  or 
picric  acid  may  be  employed.  If  the  latter,  the  freshly  extracted 
tooth  is  placed  in  a  saturated  solution  of  the  acid  containing 
two  parts  per  cent,  of  hydrochloric  acid,  and  crystals  of  picric 
acid  are  from  time  to  time  added  as  required,  the  solution 
being  stirred  frequently  with  a  glass  rod.  It  is  well  to  break 
or  cut  open  the  tooth  tirst,  so  as  to  expose  the  pulp  cavity,  if 
this  can  be  done  without  disarranging  the  contents.  When 
softened  throughout  — this  can  be  tested  by  attempting  to  pass 
a  line  needle  through  it — the  tooth  is  placed  in  spirit  (containing 
lithium  carbonate),  which  should  be  changed  until  it  ceases  to 
become  coloured  by  the  excess  of  picric  acid.  All  that  remains 
to  be  done  is  to  embed  in  paraffin  by  the  usual  method,  tix  the 
sections  to  a  slide  and  stain  with  hrematein  or  picrocarmine 
or  some  other  dye,  mounting  either  in  glycerine  jelly  or  m 
xylol  balsam. 

Study  of  decalcified  teeth  '  in  situ.' — Still  more  instructive 
preparations  are  obtained  by  softening  a  portion  of  the 
lower  jaw  with  the  teeth  iyi  situ,  and  making  sections 
through  the  whole  structure.  It  is  best  to  take  the  jaw  of 
a  small  animal — a  rat,  for  instance.  The  flesh  having  been 
cleared  away,  the  softening  is  effected  with  picric-hydro- 
chloric acid  or  with  phloi-oglucin-nitric  acid  ;  and  then,  after 
due  washing  with  lithium  carbonate  spirit,  the  piece  is 
embedded,  cut,  stained,  and  mounted  as  before. 

Besides  showing  the  teeth  and  the  way  in  which  they 
are  inserted  into  the  lower  jaw,  the  structure  of  this  bone 
is  itself  well   demonstrated.     At    the    lower   part    the   con- 


216  PRACTICAL  HISTOLOGY 

stantly  growing  incisor,  which  extends  in  the  rat  below  the 
molars  to  the  back  part  of  the  jaw,  exhibits  the  large  elon- 
gated odontoblasts  of  a  developing  tooth,  with  their  well- 
marked  dentinal  processes,  which  in  some  parts  project  like 
harp-strings  across  a  small  space,  produced  by  shrinkage,  which 
intervenes  between  the  cells  and  the  dentinal  substance. 
It  will  be  remarked,  also,  that  in  these  teeth  the  most 
newly  formed  layer  of  dentine  becomes,  especially  near  its 
junction  with  the  older  parts,  very  intensely  stained  by 
the  logwood.  This  is  the  case  with  all  teeth  which  are  still 
in  process  of  development.  Carmine  has  not  the  same  action. 
Development  of  the  teeth. — For  the  study  of  the  develop- 
ment of  the  teeth  sections  are  made  of  the  jaws  of  embryos 
and  young  animals ;  perhaps  the  most  convenient  to  choose 
are  foetal  and  new-born  rats.  Sections  of  their  jaws  exhibit  not 
only  the  mode  of  development  of  the  teeth,  but  also  that  of 
the  hair,  the  bone  of  the  lower  jaw  (which  ossifies  in  the 
connective  tissue  around  Meckel's  cartilage),  the  tongue, 
and  many  other  parts.  The  preparation  is  as  follows  : — 
The  foetuses  or  young  animals  are  decapitated,  and  the 
heads  dropped  into  0.2  per  cent,  chromic  acid  or  Flemming's 
fluid.  After  a  week's  time,  during  which  the  liquid  is  fre- 
quently stirred,  they  are  transferred  to  weak  spirit  containing 
lithium  carbonatCj  and  in  a  day  or  two  to  strong  spirit.  After 
being  in  this  for  two  or  three  days  they  are  ready  for  cutting. 
Either  the  lower  jaw  is  removed  and  embedded  separately,  or  the 
whole  head  is  embedded,  and  both  jaws  are  cut  simultaneously. 
The  sections  are  to  be  stained  with  logwood,  carmalum  or 
picro-carmine,  unless  the  preparation  has  been  stained  in 
bulk.  The  stay  in  chromic  acid  may  not  have  been  long 
enough  to  remove  all  the  earth  from  the  partly  developed 
bones  and  teeth,  but  what  still  remains  is  so  small  in 
amount  that  it  will  not  prevent  a  thin  section  being  made. 
The  earlier  stages  in  the  development  of  the  teeth  may  be 
perhaps  seen  in  the  molar  region  ;  the  later  stages,  com- 
prising the  development  of  the  dental  tissues,  especially  the 


THE   TONGUE  217 

dentine  and  enamel,  may  l)e  studied  in  the  much  more  ad- 
vanced incisors,  whicli,  as  just  pointed  out,  extend  back- 
wards in  these  animals  through  the  greater  part  of  the  length 
of  the  jaw. 

THE    TONCUE 

Sections, — Small  portions  of  this  organ  from  dillerent 
parts  are  hardened  in  3  per  cent,  bichromate  of  potash 
(fourteen  days),  and  subsequently  in  spirit,  and  are  embedded, 
so  as  to  be  cut  vertically  to  the  surface  of  the  mucous  membrane. 
The  sections  are  stained  with  ha^matein,  and  mounted  in 
balsam.  A  double  staining  with  picric  acid  and  logwood  or 
with  picro-carmine  may  also  be  employed  in  the  same  way 
as  with  the  sections  of  skin  (p.  200).  The  stratified  epithelium, 
the  papillje  of  the  mucous  membrane,  and  the  arrangement  of 
the  muscular  fibres,  as  well  as  the  mode  of  termination  of  the 
superficial  muscular  fibres  in  the  connective  tissue  of  the 
mucous  membrane,  may  be  studied  in  these  sections.  Some  of 
the  sections  include  mucous  glands,  which  differ  in  appearance 
according  to  their  condition  at  the  time  of  death.  If  they 
had  not  been  recently  stimulated  by  the  ingestion  of  food  or 
otherwise,  the  cells  of  the  gland  will  still  be  tilled  with  mucus, 
as  shown  by  their  swollen  appearance,  and  by  their  becoming 
strongly  stained  by  the  logwood  ;  whereas,  on  the  contrary, 
if  they  have  recently  discharged  their  secretion,  the  cells  will 
be  granular  and  almost  colourless,  and  there  will  be  indica- 
tions of  mucus  in  the  lumina  of  the  ducts. 

Besides  the  mucous  glands  others  may  be  seen  in  the 
neighbourhood  of  the  papilhe  vallatte,  which  secrete  no 
mucus,  and  consequently  do  not  present  the  above  differences 
of  staining.  Many  of  their  ducts,  if  the  section  pass  in  the 
direction  they  take,  will  be  found  to  open  into  the  fossa;  of  the 
circumvallate  papilla^,  or,  at  least,  near  those  parts  in  which 
taste-buds  have  been  found.  The  taste-buds  themselves  may 
possibly  be  seen  in  the  epithelium  on  the  sides  of  the  papillaj 
vallatie,  and  also  in  that  which  covers  the  mucous  membrane 


218  PRACTICAL   HISTOLOGY 

near  the  root  of  the  tongue  on  each  side.  The  description  of 
other  modes  of  preparing  them  will,  however,  be  deferred  until 
the  organs  of  special  sense  are  treated  of. 

"Vessels  of  the  tongue. — Sections  should  also  be  made  of  an 
injected  tongue.  These  will  show  not  only  the  numerous  vas- 
cular loops  in  the  more  obvious  papillae  of  the  mucous  mem- 
brane, corresponding  in  number  with  the  microscopic  secondary 
papillae,  but  also  the  arrangement  of  the  vessels  in  the  muscular 
substance  of  the  organ.  The  injected  specimens  are  much 
improved  by  slightly  staining  them  with  logwood  or  eosin. 

Palate  and  tonsils. — The  soft  palate  and  the  tonsils  may 
be  hardened  in  the  same  way  as  the  tongue,  or,  by  immersion 
in  strong  spirit.  The  sections  are  stained  and  mounted  in  the 
usual  way. 

THE    SALIVARY    GLANDS 

Sections. — These  organs  are  prepared  by  placing  small 
pieces  of  them  as  soon  after  death  as  possible  in  formol, 
sublimate,  or  picric  acid  solution,  or  in  Flemming's  or  Her- 
mann's fluids.  After  two  or  three  days  they  are  transferred 
to  spirit,  which  is  frequently  changed.  They  may  also  be 
prepared  by  merely  being  placed  in  strong  spirit  or  absolute 
alcohol  for  two  or.  three  days.  The  embedding,  staining,  and 
mounting  may  be  effected  in  the  ordinary  manner,  but 
bulk-staining  by  Heidenhain's  method  is  especially  to  be 
recommended  for  these  structures. 

Physiological  states  of  the  cells. — The  difference  in  the 
appearance  of  the  salivary  cells  previous  and  subsequent  to  the 
state  of  secretory  activity  is  best  studied  in  the  glands  of  two 
animals,  one  of  which  has  been  killed  after  some  hours'  fasting, 
the  other  a  short  time  after  administration  of  a  small  dose  of 
pilocarpin  by  hypodermic  injection.  A  small  portion  of  each 
gland  is  prepared  with  picric  acid  for  sections,  and  another  is 
treated  with  vapour  of  osmic  acid  and  afterwards  macerated 
in  water  for  a  few  days  for  teased  preparations.     Besides  these. 


SALIVARY   GLANIJS  219 

other  small  fiagineuts  arc  to  \m  teased  fresh  in  '2  percent,  salt 
solution. 

Gland  ducts  and  nerve  terminations. — The  ducts  of  the 
glands  and  their  intracellular  commencements  in  the  alveoli 
are  studied  in  sections  of  small  pieces  of  fresh  glands  prepared 
by  the  method  of  Golgi  (see  p.  152).  In  these  sections  nerve 
tenninations  may  also  be  observed. 


220  PEACTICAL  HISTOLOGY 


CHAPTER   XIY 

THE    CESOPHAGUS    AND    STOMACH 

Sections  of  oesophagus. — The  oesophagus  is  hardened  for 
the  preparation  of  sections  by  picric  acid,  0-2  per  cent,  chromic 
acid,  Flemming's  fluid,  formol,  alcohol,  or  3  p.c.  bichromate  of 
potash.  After  three  or  four  days  in  any  of  the  first  three,  the 
tissue  may,  as  usual,  be  transferred  to  spirit.  Before  putting  it 
into  the  mixture  it  should,  if  but  a  small  piece  be  employed,  be 
pinned  out  upon  a  piece  of  cork,  so  as  to  stretch  it  slightly  and 
avoid  folds.  But  if  a  tubular  piece  be  available,  this  object  may 
be  effected  more  satisfactorily  by  distending  the  organ  with  the 
preservative  solution  through  a  glass  cannula  tied  into  one  end, 
the  other  end  having  been  secured  by  a  ligature  before  the 
distension  ;  the  piece  is  then  immersed  in  the  mixture  for  a 
few  hours,  after  which  it  may  be  cut  open. 

Heidenhain's  bulk-stain  gives  good  results  with  the 
oesophagus. 

In  cutting  a  piece  of  one  of  the  membranous  viscera  it  is 
well  to  place  it,  as  a  general  rule,  so  that  the  sections  shall  be 
exactly  transverse  to  the  axis  of  the  viscus,  following,  there- 
fore, the  direction  of  the  circular  muscular  fibres  and  cutting 
the  longitudinal  across.  When  the  direction  of  the  section  is 
known,  it  is  easier  to  understand  the  appearances  which  the 
various  parts  present  when  cut.  Sections  cut  parallel  to  the 
axis  of  the  viscus,  and  taking  therefore  the  direction  of  the 
longitudinal  muscular  fibres,  may  be  made  with  nearly  equal 
advantage,  but  oblique  directions  should  be  avoided. 

Blood-vessels   of  the   gullet. — The   arrangement   of    the 


THE   STOMACH  2'21 

vessels  is  best  sliown  in  a  flat  prej:Kii'ation.  A  small  piece, 
obtained  from  a  small  injected  animal,  is  transferred,  without 
staining,  from  spirit  to  oil  of  cloves,  and  subsequently  mounted 
in  balsam,  with  the  inner  surface  uppermost.  Such  a  prepa- 
ration is  only  useful  for  examination  with  a  low  power,  but 
by  this  the  .arrangement  of  the  vessels  in  the  successive  strata 
can  be  well  made  out.  If  too  thick  to  be  mounted  as  one 
specimen,  the  mucous  membrane  and  muscular  coat  may  be 
dissected  apart  and  mounted  separately.  Vertical  sections 
of  injected  oesophagus  may  also  be  made. 

The  stomach. — -The  stomach  should  always  be  prepared 
as  soon  as  possible  after  death,  for,  in  the  tirst  place,  the 
columnar  epithelial  cells  covering  the  inner  surface  soon  become 
altered  :  and  secondly,  if  digestion  were  proceeding  in  the 
organ  at  the  time  of  death,  the  mucous  membrane  itself  be- 
comes attacked  by  the  gastric  juice  in  a  very  short  time. 

The  abdomen,  therefore,  is  to  be  opened  as  soon  as  the 
animal  (a  cat  or  dog)  is  dead,  the  o?sophagus  cut  as  near  the 
diaphragm  as  possible,  and  the  duodenum  about  two  inches 
beyond  the  pylorus  ;  the  folds  of  peritoneum  connecting  the 
viscus  to  the  liver  and  neighbouring  parts  are  also  severed, 
and  the  stomach  is  removed.  If  the  organ  is  empty  or  if  the 
contents  are  fluid  enough  to  admit  of  being  poured  out 
through  the  pylorus,  it  may  be  prepared  as  a  whole  by  dis- 
tension with  the  hardening  fluid,  which  may  be  cori-osive 
sublimate,  formol,  picric  or  chromic  acid,  followed  by  spirit. 
The  duodenal  end  is  tied  up  and  a  glass  cannula  is  fastened 
into  the  oesophageal  end.  This  is  connected  by  an  indiarubber 
tube  with  a  bottle  containing  salt  solution.  A  second  tube 
passes  through  the  cork  of  this  bottle,  and  by  blowing  through 
it  the  salt  solution  is  forced  into  the  stomach  and  washes  out 
its  contents.  The  salt  solution  is  then  replaced  by  the  harden- 
ing fluid.  When  the  organ  is  moderately  distended  witli  this 
the  indiarubber  tube  is  clipped,  to  prevent  any  of  the  liquid 
being  forced  back  into  the  bottle  Ijy  the  contraction  of  the 
muscular  walls  of  the  stomach  ;  the  lower  end  of  the  gullet  is 


222  PEACTICAL  HISTOLOGY 

then  secured  by  a  ligature,  and  the  whole  organ  is  immersed 
in  a  large  receptacle  filled  with  the  same  hardening  fluid. 
After  a  few  hours  it  should  be  opened  and  put  into  fresh 
fluid  ;  or  if  it  is  not  desired  to  keep  the  whole  of  the  organ, 
small  pieces  only  from  diflerent  parts  are  so  transferred.  If 
the  stomach  be  too  large  to  harden  as  a  whole,  or  difiicult  to 
be  cleared  of  its  contents,  small  pieces  may  be  cut  out  from 
the  fresh  organ,  pinned  out  on  a  piece  of  cork  or  cake  of 
wax,  washed  with  salt  solution,  and  then  immersed  in  the 
hardening  fluid.  In  two  or  three  days  the  tissue  is  ready 
to  be  transferred  to  spirit.  The  sections,  which  should  com- 
prise all  the  coats  of  the  organ,  are  to  be  stained  with 
carmalum,  or  with  hsematein  and  eosin,  or  with  toluidin  blue 
and  eosin,  and  mounted  in  balsam  as  usual. 

Heidenhain's  bulk-stain  may  also  be  employed  with  ad- 
vantage. 

One  set  of  sections  should  be  taken  longitudinally  through 
the  pylorus  in  order  to  show  the  manner  in  which  the  pyloric 
glands  of  the  stomach  become  continued  into  the  duodenum 
as  the  glands  of  Brunner.  Other  sections  should  pass  through 
the  junction  of  the  oesophagus  with  the  stomach. 

Gastric  glands. — For  making  out  distinctly  the  structure 
of  the  mucous  membrane  and  the  character  of  the  cells  which 
occupy  the  gastric  glands,  it  is  better  to  take  small  portions  of 
the  mucous  membrane  only.  With  this  object,  small  pieces 
of  the  fresh  mucous  membrane  are  taken  from  two  or  three 
distinct  parts — one  from  near  the  pylorus,  one  from  the 
fundus,  and  one  from  close  to  the  oesophageal  opening — and 
placed  at  once  in  absolute  alcohol  or  in  corrosive  sublimate 
or  in  picric  acid,  to  be  followed  by  alcohol.  When  hardened 
they  are  embedded  separately,  and  vertical  sections,  as  thin  as 
possible,  are  made.  Some  of  these  are  to  be  stained  with  car- 
malum and  picric  acid  and  mounted  as  before  in  balsam,  but 
one  or  two  of  the  thinnest  may  be  selected  and  mounted  in 
glycerine.  Others  may  be  placed  for  twenty-four  hours  or 
more  in  picrocarmine  solution.     Others  may  be  stained  with 


THE   STOMACH  223 

toluidin  blue  and  eosin,  and  yet  other  sections  with  Nichol- 
son's blue  No.  1,  and  mounted  in  glycerine.  Lastly,  a  small 
fragniont  from  each  part  may  be  put  fresh  into  1  per  cent, 
osmic  acid,  and  after  24  hours  washed  in  water,  embedded 
in  gum,  and  cut  frozen. 

These  different  modes  of  staining  bring  out  distinctly  the 
differences  between  the  various  kinds  of  cells  found  in  the 
glands.  In  the  logwood  preparations  the  parietal  cells  will  be 
found  stained  rather  less  than  the  rest,  whereas  in  the  sections 
stained  by  carmine,  eosin,  and  toluidin  blue  they  are  coloured 
more  deeply.  This  is  especially  the  <;ase  in  the  Nicholson-l)lue 
preparations,  where  the  peptic  cells  are  stained  of  a  deep  colour, 
whilst  the  other  cells  may  remain  almost  uncoloured.  Many 
animals  have  a  special  kind  of  glands  near  the  cardiac  orifice, 
differing  from  the  fundus-glands  in  containing  no  parietal 
cells. 

Horizontal  sections. — Besides  the  vertical  sections  of  the 
mucous  membrane,  others  are  to  be  made  parallel  with  the 
inner  surface,  and  therefore  so  as  to  cut  the  glands  across. 
The  sections  will,  of  course,  comprehend  in  succession  first  the 
mouths  of  the  glands,  then  their  necks,  and  finally  the  deeper 
parts.  They  ai'e  to  be  stained  and  mounted  in  the  same  way 
as  the  vertical  sections. 

Cells  of  the  glands,  isolated. — In  addition  to  studying 
them  in  sections  in  this  way,  the  cells  of  the  glands  may  also 
be  studied  in  teased-out  preparations  of  the  fresh  mucous 
membrane  made  in  serum  or  0"9  per  cent,  salt  solution.  The 
cells  will  be  mox"e  readily  obtained  separate  if  a  small  piece  of 
the  membrane  is  placed  in  one-third  alcohol  for  twenty-four 
or  forty-eight  hours,  but  are  apt  to  be  somewhat  altered,  and 
the  columnar  cells  of  the  general  surface  and  mouths  of  the 
glands  to  become  transformed  into  goblet-cells,  by  the 
swelling  and  escape  of  their  contained  mucus. 

Blood-vessels  of  the  stomach.— Both  vertical  and  horizon- 
tal sections  of  an  injected  stomach  may  be  made.  This  may  be 
obtained    from  the    animal  which  was  injected  entire  :  if  it 


224  PEACTICAL  HISTOLOaY 

were  a  rat,  the  preparations  are  to  be  made  from  the  pyloric 
half  of  the  organ,  since  in  this  animal  the  cardiac  end  has  a 
non-glandular  mucous  membrane  with  stratified  epithelium  like 
that  of  the  gullet.  The  vertical  sections  need  not  be  very- 
thin  ;  they  are  improved  by  being  stained  with  alcoholic  eosin, 
so  as  to  become  slightly  coloured,  before  being  mounted  in 
balsam  by  the  ordinary  process.  Instead  of  cutting  horizontal 
sections  a  small  piece  of  the  injected  stomach  may,  if  from  a 
small  animal,  be  simply  mounted  flat  with  the  inner  surface 
uppermost,  without  staining. 

Lymphatics. — An  attempt  may  be  made  to  inject  the  lymphatics 
of  the  gastinc  mucous  membrane  with  Berlin  blue,  but  the  process 
requires  considerable  care  and  experience,  since  it  presents  unusual 
difiiculties.  If  a  successful  result  is  obtained  the  injected  portions 
are  hardened  in  alcohol,  and  vertical  sections,  which  may  be 
tolerably  thick,  made  and  mounted  in  balsam. 

Nerves. — The  nerve  plexuses  and  ganglia  in  the  coats  of  the 
stomach  may  be  shown  by  the  methods  recommended  for  the  intes- 
tinal nerves  (p.  230).  The  silver  chromate  method  also  serves  to 
show  the  ductules  which  lead  from  the  lumen  of  the  fimdus  glands 
to  the  parietal  cells. 


22i 


CHAPTER  XV 

THE    SMALL    AND    LARGE    INTESTINE 

Sections  of  small  intestine. — Pieces  of  the  small  intestine 
are  prepared  in  exactly  tlie  same  way  as  the  stomach,  the  gut 
being  distended  by  the  hardening  fluid.  After  a  few  hours 
the  intestine  is  opened  and  the  fluid  changed,  and  in  three  or 
four  days  the  tissue  is  transferred  to  spirit,  to  complete  the 
hardening.  Three  pieces  of  the  small  intestine  are  to  be  pre- 
served in  this  way — viz.,  one  from  the  commencement  of  the 
duodenum  (this  will  probably  have  been  included  in  the 
stomach  preparation)  ;  a  second  from  the  jejunum  ;  and  the 
third  from  the  ileum,  including  one  of  the  patclies  of  Peyer. 
The  pieces  may  be  obtained  from  a  cat,  dog,  or  rabbit,  the 
contents  of  the  intestine  being  first  cleared  out  by  gently 
squeezing  the  gut,  or  by  forcing  a  rapid  stream  first  of  salt 
solution  and  then  of  the  hardening  fluid  through  each  piece 
before  tying  up  the  further  end.  Instead  of  distending  it 
with  the  preservative  fluid,  the  gut  may  be  opened  and  kept 
in  an  extended  state  by  pinning  it  on  a  cork  or  cake  of 
wax,  which  is  then  inverted  into  the  fluid. 

It  is  necessary,  in  order  to  see  the  structure  of  the  villi,  that 
the  sections  should  be  very  thin  so  as  to  include  not  the  whole 
thickness  of  a  villus,  but  only  a  longitudinal  slice,  otherwise 
the  epithelium  on  its  surfaces  interferes  with  the  view  of  the 
internal  structure.  One  piece  must  be  so  cut  as  to  obtain 
sections  aci'oss  the  villi,  which  is  easily  done  with  paraflin-em- 
bedding,  the  sections  being  fixed  to  the  slide  in  the  usual  way. 

Fat  absorption. — ^For  the  purpose  of  studying  the  course 


226  PEACTICAL  HISTOLOGY 

which  fatty  particles  take  in  passing  from  the  cavity  of 
the  intestine  into  the  central  lacteals  of  the  villi,  a  rat  is 
killed  three  or  four  hours  after  a  meal  containing  fat.  On 
opening  the  abdomen  the  lacteals  in  the  mesentery  should  be 
found  filled  with  chyle,  and  the  cavity  of  the  small  intestine 
occupied  by  emulsified  fat  which  is  undergoing  absorption. 
The  intestine  is  opened  at  once,  and  two  or  three  very  small 
pieces  of  the  mucous  membrane  are  snipped  ofi"  and  treated 
with  1  per  cent,  osmic  acid  or  osmic  acid  vapour  for  an  hour. 
Another  minute  piece  is  quickly  teased  out  with  needles  in 
a  drop  of  serum  or  salt  solution  ;  a  piece  of  hair  is  added, 
and  the  preparation  is  covered  and  examined.  One  of  the 
portions  treated  with  osmic  acid  is  allowed  to  remain  forty- 
eight  hours  in  water,  and  is  then  broken  up  with  needles  and 
by  tapping  the  cover-glass.  Another  is  placed  in  gum,  and 
when  permeated  with  this  sections  are  cut,  frozen,  and 
mounted  in  glycerine  or  glycerine  jelly. 

In  the  two  teased  preparations — serum  and  osmic — many 
of  the  columnar  epithelium  cells  will  be  found  to  contain  fatty 
globules  of  various  sizes  (stained  black  in  the  osmic  prepara- 
tion). Similar,  but  for  the  most  part  smaller,  particles  will 
also  be  found  in  some  of  the  numerous  lymph  corpuscles  which 
are  set  free  from  the  retiform  tissue  of  the  mucous  membrane 
by  the  process  of  teasing.  In  the  sections  the  epithelium  cells 
and  the  lymph  corpuscles  will  be  observed,  in  situ,  in  the  same 
condition,  viz.  containing  blackened  fatty  particles,  and  more- 
over the  cleft-like  central  lacteal  in  the  middle  of  each  villus 
may  be  found  to  contain  not  only  similar  globules,  but  also 
lymph-cells  in  process  of  disintegration.  Hence  we  infer  that 
the  fatty  matters  are  first  taken  up  from  the  cavity  of  the 
intestine  by  the  columnar  epithelium  cells  ;  that  they  are 
transmitted  from  these  to  the  interstitial  tissue  of  the  villus, 
where  they  are  taken  up  by  amoeboid  lymph-cells,  and  that 
these  convey  them  into  the  central  lacteal.  Where  absorption 
is  proceeding  very  rapidly,  some  of  the  fatty  particles  may  be 
found  in  the  interstices  of  the  connective  tissue  of  the  villus, 


SMALL  INTESTINE  227 

not  included  within  leucocytes  ;  and  it  is  probable  that  under 
these  circumstances  some  of  the  fat  may  pass  into  the  lacteals 
independently  of  tliose  cells.  This  is  especially  the  case  in  the 
dog,  but  in  other  animals,  such  as  the  rat  and  guinea-pig,  the 
whole  of  the  fat  is  usually  included  within  either  the  epi- 
thelium cells  or  the  leucocytes,  and  only  becomes  free  within 
the  lacteals.  Similar  observations  may  be  made  on  a  frog  killed 
two  days  after  being  fed  with  lard. 

Vessels  of  the  small  intestine. — The  bhod-vnssels  of  the 
small  intestine  are  studied  in  vertical  sections  of  the  injected 
gut.     The  sections  may  be  stained  with  eosin. 

The  lymphatics  (lacteals)  may  perhaps  be  seen  in  thin 
sections  of  the  uninjected  preparations  as  cleft-like  spaces  in 
the  villi  and  in  the  substance  of  the  mucous  membrane,  and 
surrounding  the  bases  of  the  lymphoid  nodules  which  make 
up  the  Peyerian  patches.  It  is  not  an  easy  matter  to  inject 
those  of  the  mucous  membrane,  although  the  larger  plexuses 
of  the  submucous  and  muscular  coat  can  be  more  easily 
demonstrated  in  this  way. 

Nerves  of  the  intestinal  wall. — The  nerves  of  the  intes- 
tinal canal  form  a  very  intei'esting  subject  of  study,  compris- 
ing some  of  the  closest  and  most  richly  gangliated  plexuses  of 
fibres  which  are  met  with  in  the  sympathetic  system.  They 
may,  moreover,  by  the  use  of  the  chloride  of  gold,  the  chromate 
of  silver,  or  the  methylene  blue  methods,  be  shown  in  all 
parts  of  either  the  small  or  the  large  intestine.  It  is  prefer- 
able to  choose  an  animal  {e.g.  rabbit  or  guinea-pig)  in  which 
the  intestinal  coats  are  thin.  The  following  is  the  mode 
of  procedure  for  the  chloride  of  gold  method  : — A  piece 
of  glass  tubing  about  a  quarter  of  an  inch  in  diameter  and 
five  or  six  inches  long  is  taken  and  one  end  is  drawn  out  into 
a  cannula,  whilst  to  the  other  a  small  piece  of  indiarubber 
tube,  furnished  with  a  spring  clip,  is  attached.  Chloride  of 
gold  solution  {\  per  cent.)  is  drawn  up  into  the  glass  tube  so  as 
almost  to  fill  it,  and  the  clip  is  then  closed,  to  prevent  the 
escape  of  the  fluid.     Care  should  be  taken  not  to  suck  any  of 

q2 


228  PEACTICAL  HISTOLOGY 

the  gold  solution  into  the  mouth.  A  piece  of  intestine  about 
three  inches  long  is  removed  from  the  dead  animal,  and  if 
not  already  empty  its  contents  are  washed  out  by  a  stream  of 
salt  solution.  The  intestine  thus  emptied  and  cleaned  is 
ligatured  firmly  at  one  end,  whilst  into  the  other  is  tied  the 
cannulated  end  of  the  glass  tube  containing  the  gold  solution. 
When  thus  secured  the  clip  is  opened  and  the  fluid  is  allowed 
to  flow  into  and  distend  with  moderate  force  the  piece  of  gut, 
the  action  of  gravity  being  assisted  by  gently  blowing  through 
the  indiarubber  tube.  As  soon  as  the  intestine  is  filled  with 
the  gold  solution  the  clip  is  again  allowed  to  close,  and  then^ 
while  an  assistant  holds  the  glass  tube  in  a  vertical  position, 
the  operator  ligatures  the  gut  just  beyond  the  end  of  the 
cannula,  which  may  now  be  cut  away.  The  piece  of  intes- 
tine, thus  filled  with  the  gold  solution,  is  immersed  for  half 
an  hour  in  more  in  the  same  liquid.  It  is  then  placed  in  a 
dish  of  water  and  cut  open  longitudinally  with  scissors,  so  as 
to  allow  the  contained  fluid  to  escape,  after  which  the 
puckered  ligatured  ends  may  also  be  removed.  The  tissue 
being  hardened  by  the  gold  solution,  the  piece  of  gut  which 
remains  retains  its  cylindrical  shape.  It  is  well  to  halve  it 
by  another  longitudinal  cut,  so  that  both  inner  and  outer 
surfaces  may  be  freely  exposed  to  the  light.  The  pieces  are 
now  placed,  with  their  outer  surfaces  uppermost,  in  a  glass 
vessel  of  water  containing  just  enough  acetic  acid  to  be  sour 
to  the  taste,  and  the  vessel  is  covered  and  allowed  to  stand 
in  a  warm  place  freely  exposed  to  the  sunlight  (see  p.  120). 
After  two  days  its  colour  will  be  found  to  have  changed  to  a 
dark  violet.  A  little  methylated  spirit  may  then  be  added  ta 
the  fluid  ;  this  serves  to  aid  the  reduction  of  the  gold  and  to 
prevent  the  growth  of  fungi.  In  another  day  or  two  the 
tissue  will  be  so  dark  as  to  appear  almost  black.  A  portion 
is  then  removed  to  a  glass  dish  of  water  and  prepared  in  the 
following  way.  In  the  first  place,  the  glandular  mucous  mem- 
brane is  sepai'ated  from  the  rest  of  the  intestinal  wall,  either 
by  tearing  it  off  with  forceps  or  by  scraping  it  away  with  the 


THE   SMALL   INTESTINE*  229 

end  of  a  blunt  scalpel.  There  now  remain  tlie  serosa  and 
two  niuscular  layers,  together  with  the  submucosa.  To  the 
inner  surface  of  the  latter  the  nmscularis  mucosjf  will  be 
still  adherent.  The  separated  fragments  of  tlie  n\ucous  mem- 
brane are  got  rid  of  by  pouring  away  the  water  first  used  and 
substituting  fresh,  and  then  an  attempt  must  be  made,  by 
aid  of  two  pairs  of  forceps,  to  peel  the  suliiuucosa  oH"  from 
the  inner  surface  of  the  muscular  coat.  Of  course  if  the  mus- 
cularis  mucosae  has  been  left,  that  will  form  a  part  of  the 
layer  which  is  thus  removed.  The  separation  must  be  done 
slowly  and  carefully,  so  as  to  get  as  large  a  piece  as  possible 
intact.  When  this  is  accomplished  satisfactorily  a  slide  is 
immersed  in  the  water,  and  the  portion  of  submucosa  so  de- 
tached is  floated  on  to  it,  and  removed  from  the  water.  Its 
further  preparation  consists  in  allowing  the  excess  of  water 
to  run  off,  applying  a  cover-glass,  making  sure  first  of  all  that 
the  layer  is  free  from  folds,  and  then  allowing  glycerine  to  pass 
under  the  cover-glass  and  replace  the  water  as  this  evaporates. 
Returning  to  the  remainder  of  the  piece  of  intestine,  the 
next  process  consists  in  picking  away  bit  by  bit  with  forceps 
the  comparatively  thick  layer  of  circular  muscular  fibres. 
This  is  not  a  difficult  proceeding,  and  when  it  is  finished  all 
that  I'emains  is  the  thin  serous  coat  and  the  longitudinal 
muscular  layer,  to  the  inner  side  of  which  the  nervous  plexus 
of  Auerbach,  the  intermuscular  plexus,  is  adherent.  No 
further  separation  is  required,  all  that  is  necessary  being  to 
float  the  piece  of  tissue  on  to  a  slide  with  the  (concave)  inner 
surface  uppermost.  But  before  applying  the  cover-glass  the 
preparation  is  to  be  examined  with  a  low  power,  to  see  that 
the  surface  of  the  serous  membrane  is  free  from  a  finely 
granular  precipitate  which  is  apt  to  be  deposited  in  the 
acidulated  water.  If  this  is  present,  the  piece  must  be  re- 
placed in  the  water  and  the  precipitate  gently  brushed  ofF 
with  a  soft  camel-hair  pencil.  The  jireparation  is  completed 
in  the  same  way  as  that  of  the  submucosa.  The  latter  shows 
Meissner's   plexus,    the  cords  of  which  are  much  finer  than 


230  •  PEACTICAL  HISTOLOGY 

those  of  Auerbach's.  In  both  plexuses  the  nervous  cords  are 
stained  of  a  violet  colour  by  the  reduction  of  the  gold  ;  at  the 
points  of  junction  of  the  nervous  cords  are  groups  of  small 
ganglion  cells,  the  nuclei  of  which  are  hardly  stained  at  all, 
and  consequently  look  clear  in  the  midst  of  the  darkly-stained 
cell-bodies.  The  distinction  between  the  individual  cells  is 
difficult  to  make  out.  Branches  may  perhaps  be  traced  pass- 
ing from  the  plexus  of  Auerbach  amongst  the  muscular  fibre- 
cells  :  from  that  of  Meissner  to  the  muscularis  mucosae,  if  this  is 
present,  and  perhaps  also  to  the  small  blood-vessels,  which  are 
particularly  well  seen  in  the  preparation  of  the  submucous  coat. 

This  is  Cohnheim's  method,  but  Lowit's  modification  may 
also  be  used.  In  this  process  the  pieces  of  gut  after  being  re- 
moved from  the  chloride  of  gold  solution  are  transferred  to 
formic  acid  solution  (1  in  4)  and  kept  in  the  dark  for  twenty- 
four  hour's  or  more,  until  the  reduction  is  complete. 

For  the  chromate  of  silver  method  very  small  pieces  of  the 
intestine  are  taken  and  treated  as  described  on  p.  152.  From 
these  sections  are  cut  and  are  mounted  in  balsam  without  a 
cover-glass. 

For  the  methylene  blue  method  a  solution  of  the  dye  in 
500  parts  normal  saline  is  used  to  distend  the  piece  of  gut, 
which  is  then  tied  up  and  immersed  in  some  of  the  same 
solution  for  about  an  hour.  It  is  then  opened,  washed  with 
normal  saline,  and  placed  in  Bethe's  fluid  (see  p.  160).  After 
the  stain  is  fixed  by  means  of  this,  the  nerves  can  be  investi- 
gated either  in  flat  preparations  or  in  sections,  mounted  in 
xylol  balsam. 

Large  intestine. — For  hardening  the  tissue  and  preparing 
sections  of  the  large  intestine  the  same  methods  are  employed 
a5  for  the  small  intestine,  so  that  it  is  unnecessary  to  re- 
capitulate them. 

The  injected  large  intestine  is  prepared,  like  the  stomach,, 
by  means  of  vertical  and  horizontal  sections. 

The  lymphatics  are  not  easy  to  inject,  but  present  less 
difficulty  than  those  of  the  stomach. 


231 


CHAPTER   XVI 

THE    LIVER    AND    PANCREAS 

TTninjected  liver. — To  prepare  sections  of  the  liver  small 
pieces  are  placed  in  3  per  cent,  bichromate  of  potash  solution 
for  ten  days,  transferred  from  this  to  weak  spirit,  and  in 
twenty-four  hours  are  placed  in  strong  spirit,  to  complete  the 
process  of  hardening.  Chromic  acid  (0-2  per  cent.),  picric  acid 
(saturated),  corrosive  sublimate,  and  formol  (5  per  cent.),  may 
all  be  used  instead  of  bichromate  of  potash,  but  much  smaller 
pieces  must  be  taken,  except  for  formol.  They  will  also  be 
ready  to  transfer  to  spirit  in  a  much  shorter  time.  The  sec- 
tions are  stained  by  the  usual  methods,  and  mounted  in 
balsam.  They  should  be  made  in  two  directions,  viz.  (1) 
in  a  plane  near  and  parallel  to  one  of  the  surfaces  of  the 
liver,  and  (2)  vertical  to  the  surface.  Those  made  in  the 
direction  first  named  will  for  the  most  part  cut  the  central  or 
intralobular  veins  across,  those  in  the  second  direction  may 
take  them  along  their  length  ;  the  apparent  arrangement  of 
the  blood  capillaries  and  liver  cells  in  the  individual  lobules 
will  differ,  both  in  accordance  with  this  difference  of  direc- 
tion and  also  according  as  the  lobule  is  cut  exactly  through 
its  centre  or  at  some  part  more  or  less  removed  from  this. 
Between  the  lobules  are  seen  the  branches  of  the  portal 
vein,  always  accompanied  by  a  branch  of  the  bile  duct,  the 
columnar  epithelium  of  which  is  very  well  seen  in  these  pre- 
parations, and  by  a  branch  of  the  hepatic  artery.  All  three  are 
included  in  a  mass  of  connective  tissue,  a  prolongation  of 
Glisson's  capsule,  enclosing  them  in  a  so-called  portal  canal. 


232  PEACTICAL  HISTOLOGY 

In  this  connective  tissue  cleft-like  spaces  may  generally  be 
seen — two  or  three  in  the  section  of  a  portal  canal — not  merely 
breaks  in  the  connective  tissue,  but  with  quite  a  definite  wall. 
These  are  the  accompanying  lymphatics.  Other  lymphatics 
accompany  the  branches  of  the  hepatic  veins,  but  are  not 
so  easily  seen  in  the  sections,  although  they  can  be  in- 
jected. The  branches  of  the  hepatic  veins  are  readily  dis- 
tinguished from  those  of  the  portal  vein,  by  the  fact  that 
they  run  unaccompanied  by  branches  of  the  bile  duct  and 
hepatic  artery.  The  blood-capillaries  of  the  lobules  look  like 
spaces  (filled  with  round  clear  bodies,  the  altered  blood 
corpuscles)  between  the  rows  of  cells  (in  the  sections  these 
appear  arranged  simply  in  rows)  ;  their  walls  are  very  thin, 
and  the  hepatic  cells  appear  for  the  most  part  to  come  in  con- 
tact with  the  wall.  But  in  reality  there  is  a  second  delicate 
membrane  around  many  of  the  capillaries,  and  between 
it  and  the  wall  of  the  vessel  is  a  space  for  the  passage  of 
lymph  (perivascular  lymphatic)  ;  it  is  difficult  to  make  this 
out,  however,  in  preparations  in  which  the  lymphatics  are  not 
injected.  The  round  nuclei  of  the  liver  cells  are  deeply 
stained  by  the  logwood,  and  the  cells  themselves  slightly.  In 
the  thinnest  parts  of  the  sections  the  lines  of  junction  between 
neighbouring  cells  can  be  well  made  out,  and  not  unfrequently 
the  small  capillary  passage  for  the  bile  which  intervenes  be- 
tween the  adjacent  sides  of  the  cells  can,  according  to  the 
direction  in  which  it  runs,  be  recognised  with  a  very  high 
power  either  as  a  horizontal  line  or  as  a  minute  aperture. 
To  obtain  the  best  results  the  pieces  of  liver,  which  are  not  to 
be  more  than  an  inch  or  so  square  and  a  quarter  of  an  inch 
thick,  should  be  placed  in  the  bichromate  solution  quite  fresh, 
from  an  animal  killed  only  a  short  time  previously. 

Injected  liver. — The  vessels  of  the  liver  seldom  get  com- 
pletely filled  when  the  rest  of  the  body  is  injected  from  the  aorta. 
It  is  generally  necessary  to  make  a  special  injection  of  this 
organ  from  the  portal  vein.  For  this  purpose  the  usual  red  or 
blue  gelatine  injection  is  used,  the  apparatus  being  arranged 


THE    LIVER  233 

ns  described  at  p.  1 79.  The  operation  i.s  conducted  as  follows  : — 
The  animal  (rabbit)  having  been  killed  by  bleeding,'  tlie 
thorax  is  opened  ;  and  the  pericardium  being  torn  away,  the 
heart  is  raised  and  two  thread  ligatures  are  passed  round  the 
inferior  vena  cava.  One  of  these  is  tightened  as  near  the 
heart  as  possible,  and  then  a  snip  is  made  in  the  vein,  so  as 
to  allow  the  blood  to  escape  freely.  Next,  the  abdomen  is 
opened,  and  the  intestines  and  stomach  being  gently  drawn 
to  the  left  si(ie,  the  pei-itoneum  at  the  back  of  the  abdomen  is 
torn  through,  and  a  ligature  jilaced  around  the  vena  cava 
above  the  accession  of  the  renal  veins.  The  portal  vein  is 
then  found  in  the  fold  of  peritoneum  which  connects  the 
under  surface  of  the  liver  with  the  stomach,  and  a  ligature,  in 
the  noose  of  which  the  hepatic  artery  may  be  included,  having 
been  passed  round  it  near  the  liver,  a  snip  is  made  in  the 
vessel  and  the  injecting  cannula  is  tied  in.  This  cannula  is 
now  filled  by  moans  of  a  pipette  with  warm  salt  solution,  and 
the  supply  tube  (from  the  injecting  bottle)  having  been  com- 
pletely filled  by  the  injecting  fluid  to  the  exclusion  of  air,  in 
the  same  way  as  previously  described  (p.  182),  is  slipped  over 
the  open  end,  and  the  injection  at  once  allowed  to  flow.  As 
it  passes  by  the  portal  system  of  veins  through  the  lobules  of 
the  liver  into  the  hepatic  system,  it  forces  whatever  blood  is 
still  contained  in  the  bloodvessels  of  the  organ  out  into  the 
vena  cava,  whence  it  can  freely  escape  into  the  thorax  through 
the  snip  which  was  there  made  in  the  vein.  As  soon  as  all 
the  blood  is  thus  driven  out,  and  only  pure  injecting  fluid 
begins  to  pass,  this  vein  is  occluded  near  the  diaphragm  by 

'  In  injecting  the  whole  body  it  was  recommended  to  kill  the  animal  by 
chloroform.  This  was  for  the  purpose  of  having  the  blood-vessels  as  much 
dilated  as  possible.  When  an  animal  is  killed  by  bleeding,  the  arteries  con- 
tract very  considerably,  and,  remaining  contracted  some  little  time  after 
death,  offer  a  considerable  resistance  at  first  to  the  passage  of  the  injection, 
and  this  may  tend  to  spoil  the  result.  In  the  liver,  however,  the  case  is 
different,  since  it  is  not  injected  through  arteries,  but  through  veins, 
which  possess  little  contractility.  Any  blood  which  remains  in  the  vessels 
does  not,  so  long  as  it  remains  fluid,  impede  the  passage  of  the  injection,  but 
is  driven  before  it. 


234  PKACTICAL  HISTOLOGY 

the  second  thread.  The  pressure  in  the  injecting-bottle  is 
then  slowly  raised,  but  should  not  even  at  the  utmost  exceed 
three  inches  of  mercury,  for  this  amount  of  pressure  will  cause 
all  the  blood-vessels  to  be  quite  fully  distended,  and  will  effect 
a  very  considerable  consequent  enlargement  of  the  organ  ; 
more  might  cause  rupture  and  extravasation.  After  the  lapse 
of  a  few  minutes,  to  allow  of  the  complete  filling  of  all  the 
blood-vessels,  a  second  ligature  is  tied  round  the  portal  vein 
close  to  the  liver  to  prevent  the  return  of  the  still  fluid  injec- 
tion, and  the  cannula  is  cut  out  from  the  portal  vein  (the  pres- 
sure in  the  apparatus  having  first  been  removed),  and  the  body 
put  into  a  cold  place  so  as  to  permit  the  gelatine  to  solidify. 
The  process  may  be  hastened  by  pouring  cold  water — iced 
if  possible — over  the  liver.  When  the  injecting  material  is 
entirely  set,  the  organ  is  removed  and  cut  into  small  pieces? 
which  are  placed  in  spirit  for  twenty-four  hours  or  more,  or 
in  3  p.c.  bichromate  of  potash  for  a  few  days.  When  hardened 
enough,  sections  may  be  made  (in  two  directions  as  with  the 
uninjected  organ)  and  mounted,  after  passing  through  alcohol 
and  oil  of  cloves,  in  balsam. 

During  the  whole  process  of  injection  the  greatest  care 
must  be  taken  not  to  handle  the  liver  more  than  can  possibly 
be  helped,  for  it  is  very  readily  scratched  or  ruptured  and  any 
such  accident  tends  to  permit  the  escape  of  the  fluid  injec- 
tion. This  warning  applies  with  equal,  if  not  greater,  force 
to  the  operation  next  to  be  described — that,  namely,  of  filling 
the  bile-ducts. 

Bile-ducts. — The  bile-ducts  are  injected  with  Berlin  blue 
solution,  2  per  cent.,  the  mercury  apparatus  (fig.  56)  being 
used.  A  simple  syringe  will,  however,  answer  the  purpose. 
The  solution,  although  fluid  in  the  cold,  should  nevertheless 
be  employed  warm,  as  it  tends  to  flow  more  freely.  A  rabbit 
is  killed  by  bleeding,  the  abdomen  opened,  and  the  common 
bile-duct  sought  for  close  to  the  portal  vein  ;  a  ligature  is 
passed  round  the  duct  and  a  small  piece  of  card  being  placed 
under  as  a  support  and  to  separate  it  from  the  accompanying 


TIIF,    LIVKK  235 

vessels,  a  snip  is  made  into  it,  and  a  glass  cannula  is  inserted, 
and  having  been  passed  along  the  duct  as  near  to  the  liver  as 
possible,  is  tied  in.  The  cystic  duct  is  ligatured  to  prevent  the 
injection  from  passing  into  the  gall  bladder.  In  the  next 
place  the  cannula  is  tilled  with  warm  Berlin  blue  solution  by 
means  of  a  line  pipette  ;  the  (previously  tilled)  supply  tube  is 
attached,  the  clip  on  this  opened,  and  the  pressure  gradually 
raised.  The  blue  fluid,  driving  whatever  bile  there  happens 
to  be  left  in  the  ducts  before  it  into  the  lobules,  penetrates 
first  into  the  interlobular  bile-ducts,  and  from  these  into  the 
outer  parts  of  the  lobules,  forcing  the  bile  more  and  more 
towards  the  centre  ;  here  of  course  there  is  no  escape  for  it, 
except  that  a  little  may  pass  into  the  lymphatics  and  blood- 
vessels through  their  walls.  Hence  it  will  be  understood  that 
the  injection  can  only  be  made  to  fill  the  intercellular  biliary 
passages  in  the  outer  part  of  each  lobule.  The  injection  should 
be  persevered  with  for  about  half  an  hour  ;  the  bile-duct  may 
then  be  tied  and  the  injecting  apparatus  removed,  after  which 
the  liver  is  cut  out  entire,  without  injuring  it  in  any  way,  and 
placed  in  strong  spirit.  In  three  or  four  hours  it  is  cut  into 
pieces,  and  the  spirit  changed,  and  in  less  than  a  week  the 
pieces  will  be  hard  enough.  The  sections  may  be  stained 
slightly  with  logwood  or  eosin. 

Method  of  Golgi. — The  bile-ducts  are  extremely  well  shown 
by  this  method,  which  must  be  applied  to  small  pieces  of  the 
liver  in  the  manner  described  on  p.  152.  Nerve  terminations 
may  also  be  seen  in  this  preparation,  and  the  reticular  con- 
nective tissue  of  the  organ  is  also  sometimes  very  well  displayed. 

Lymphatics  of  the  liver. — The  lymphatics  of  the  liver  are  injected 
through  a  fine  cannula  stuck  obliquely  into  the  supei'ficial  j'jart  of 
the  organ  immediately  beneath  the  capsule.  Either  solution  of 
Berlin  blue  or  alkanet  may  be  used.  The  part  should  be  quite 
fresh.  If  the  injection  be  persisted  in  for  a  long  while,  the  fluid 
maj'  flow  out  both  b}'  the  lymphatics  accompanying  the  portal  vein 
and  those  accompanying  the  hepatic  veins  (Ludwig  and  Fleisehl) 
Very  frequently,  however,  the  injecting  fluid  finds  its  way  into  the 
blood-system  instead  of  the   lymphatics.      The   injection   of  the 


236  PEACTICAL  HISTOLOGY 

lymphatics  may  be  accomplished  in  another  manner,  viz.  by  seek- 
ing the  lymphatics  which  accompany  the  hepatic  veins  at  the  back 
of  the  liver,  and  tying  a  cannula  into  one  of  them.  After  a  time  the 
fluid  will  be  found  to  pass  out  by  the  vessels  which  accompany  the 
portal  vein. 

Hepatic  cells. — In  addition  to  what  may  be  learnt  from 
sections  of  the  organ,  teased-out  preparations  afford  much 
useful  information,  both  of  the  characters  of  the  liver  cells  and 
of  the  connective  tissue  of  the  lobules.  For  this  purpose  small 
portions  of  the  perfectly  fresh  and  warm  liver  are  broken  up 
in  serum  or  salt  solution,  and  other  portions  are  macerated 
for  a  day  or  two  in  one- third  alcohol,  and  subsequently  teased 
out  in  water,  and  stained  with  dilute  hsematein. 

Demonstration  of  glycogen  in  the  hepatic  cells. — This  is 
most  readily  shown  microscopically  in  the  liver  of  a  rabbit 
which  has  been  fed  a  few  hours  previously  with  a  meal  of 
carrots.  The  animal  having  been  killed  rapidly,  by  bleeding 
or  otherwise,  small  pieces  of  the  liver  are  thrown  into  strong 
spirit,  and  left  in  this  until  suflBciently  hardened.  Thin  sec- 
tions are  then  cut  by  the  paraffin  method,  and  are  treated, 
after  being  passed  through  xylol  and  absolute  alcohol,  with  a 
solution  of  iodine  in  iodide  of  potassium,  which  stains  the 
cells  which  contain  glycogen  of  a  reddish  brown  colour.  The 
sections  may  either  be  mounted  in  diluted  glycerine  or  in 
acetate  of  potash  solution.  Alcohol,  xylol,  chloroform,  &c. 
rapidly  extract  the  colour. 

Demonstration  of  iron  in  the  hepatic  cells. — This  can  be 
effected  by  the  employment  of  Macallum's  method  (p.  24).  It 
will  be  found  necessary  to  treat  with  hydrochloric  acid  prior 
to  hsematoxylin  in  order  to  exhibit  the  iron  here. 


The  pancreas  is  prepared  in  the  same  manner  as  the 
salivary  glands,  to  the  description  of  which  the  student  is 
referred.  The  zymogen  granules  are  well  shown  in  sections 
double-stained  with  carmalum  or  ha^malum  and  picric  acid  ; 
and  these  methods  of  staining  also  bring  out  well  the  peculiar 
tissue  of  the  '  islands.' 


237 


CHAPTER  XVII 

TIIK    DUCTLESS    GLANDS 

Ductless  glands  of  the  larynx  and  trachea. — The  thyroid 
and  thymus  are  studied  chiefly  by  means  of  sections,  for 
facilitating  the  preparation  of  which  the  glands  are  hardened 
in  alcohol  alone,  or  in  sublimate,  picric  acid,  or  formol,  followed 
by  alcohol.  Unless  from  a  small  animal  they  should  not  be  put 
entire  into  the  fluid,  but  cut  into  thin  pieces,  so  that  the  pre- 
servative fluid  may  penetrate  rapidly.  They  are  best  stained  by 
hiumatein,  which  colours,  besides  the  nuclei  of  the  cells,  the 
so-called  '  colloid '  which  is  met  with  in  the  vesicles  of  the 
thyroid. 

The  concentric  corpuscles  of  Hassall  which  are  met  with 
in  the  thymus  can  be  seen  in  sections  of  that  organ,  but 
may  also  be  studied  isolated  in  preparations  of  the  fresh  gland 
teased  out  in  salt  solution.  They  are  stained  yellow  by  picric 
acid.  The  pituitary  body  and  pineal  gland  are  prepared  in 
the  same  way  as  the  thymus  and  thyroid. 

Sections  of  lymphatic  glands. — These  are  chiefly  studied 
by  means  of  sections.  They  are  hardened  in  strong  spirit, 
into  which  they  are  put  immediately  after  removal  from  the 
animal  ;  in  two  or  three  days  they  are  sufficiently  tirm  to  cut, 
but  improve  if  left  longer.  Or  they  may  flrst  be  injected  inter- 
stitially  with  dilute  chromic  acid  (0-2  per  cent,)  and  placed  in 
a  quantity  of  the  same  fluid  for  a  few  days,  then  trans- 
ferred to  50  per  cent,  spirit,  which  is  to  be  changed  in  two  or 
three  days  to  strong  alcohol.  They  may  either  be  cut  from 
paraffin  or  soaked  in  gum  and  cut  by  the  freezing  method. 


238  PEACTICAL  HISTOLOGY 

The  sections,  which  must  be  very  thin  indeed,  and  should 
include  both  cortical  and  medullary  substance,  may  be  mounted 
in  glycerine,  without  staiaing,  or  they  may  be  stained  with 
hsematein  and  mounted  in  xylol  balsam.  If  the  lymph -paths 
appear  filled  up  with  lymph  corpuscles,  so  that  the  retiform 
tissue  which  traverses  them  is  not  well  seen,  but  the  whole 
section  appears  more  or  less  uniform  in  structure,  these 
corpuscles  may  be  in  great  measure  removed  by  vigorously 
shaking  up  the  sections  with  water  in  a  test-tube,  or  by 
gently  brushing  them  under  spirit  with  a  soft  camel-hair 
pencil.  Unfortunately  both  these  methods  tend  to  break  up 
the  sections,  and  indeed  it  is  not  necessary  to  employ  them 
if  the  sections  are  made  suflBciently  thin. 

The  glands  may  also  be  stained  in  bulk,  a  thin  piece  being 
placed  in  carmalum  or  some  other  good  bulk-stain  for  24  hours 
or  more ;  then  either  soaked  in  gum  and  cut  frozen  or 
embedded  in  parafiin,  and  the  sections  fixed  on  the  slide  and 
mounted  in  balsam  in  the  usual  way. 

Reticular  tissue  of  lymphatic  glands. — This  may  be  made 
manifest  in  various  ways.  One  of  the  simplest  is  to  stain  a 
section  with  acid  fuschin  dissolved  in  alcohol,  then  clear  with 
oil  of  cloves  and  mount  in  xylol  balsam.  Or  a  piece  of  fresh 
gland  may  be  frozen  and  sections  cut,  and  dipped  for  a  minute 
in  a  1  per  cent,  solution  of  caustic  potash,  then  thoroughly 
washed,  stained  with  acid  fuschin,  and  mounted  as  before.  The 
methyl-blue  and  eosiri  stain  recommended  on  p.  23  will  also 
be  found  valuable  for  differentiating  the  fine  retiform  tissue, 
since  this,  like  other  connective  tissue,  becomes  stained  of  an 
intense  blue  by  that  combination. 

The  retiform  tissue  may  also  be  well  seen  in  preparations 
made  by  Golgi's  method  (p.  152).  The  lymphatic  glands  of 
the  dog  may  be  recommended  for  demonstrating  the  structure 
of  these  organs,  especially  for  showing  the  trabeculse  and 
lymph-sinuses.  In  some  animals  the  trabeculse  are  far  less 
developed,  and  the  gland  is  little  but  a  mass  of  lymphoid 
tissue  intersected  by  lymph -channels. 


THE   SPLEEN  239 


TIIR    SPLEEN 

The  uninjected  spleen.  The  spleen  is  Iiardened  in  the 
same  manner  as  the  liver.  The  sections  are  to  be  stained  with 
hppmatoin  and  eosiji  or  carmalum  and  picric  acid,  and  mounted 
by  the  ordinary  modes  of  procedure. 

In  these  preparations  the  Malpighian  corpuscles  (or  nodules 
of  lymphoid  tissue)  are  very  strongly  coloured  by  hajmatein  ; 
the  trabecular  which  traverse  the  pulp,  which  are  largely  com- 
posed of  plain  muscular  tissue,  are  stained  yellow  by  the  picric 
acid  ;  the  substance  of  the  pulp  is  but  slightly  coloured  by  the 
logwood,  only  the  cell-nuclei,  and,  to  a  much  less  extent,  the 
network  of  the  retiform  tissue  being  stained.  The  prevailing 
colour  of  the  pulp  is  yellowish,  owing  to  the  blood,  which 
at  the  time  of  death  remained  in  the  interstices  of  the  tissue 
becoming  stained  by  the  picric.  Here  and  there  a  small 
mass  of  coarsely-granular  reddish-yellow  pigment  may  be 
detected,  lodged  in  one  of  the  corpuscles  of  the  spleen  pulp. 
With  a  high  power  the  manner  in  which  the  veins  open  out  of 
the  interstices  of  the  pulp  may  be  made  out. 

Irrigated  spleen. — By  another  mode  of  preparing  the  spleen 
all  the  blood  is  first  washed  out  by  a  stream  of  salt  solution, 
injected  through  the  splenic  artery,  and  the  salt  solution  is 
followed  by  a  stream  of  3  p.c.  bichromate  or  picric  acid.  This  is 
made  to  distend  the  organ  somewhat,  the  distension  being  main- 
tained by  ligaturing  the  vessels  near  the  hilum.  The  organ  is  now 
placed  entire  in  a  quantity  of  the  solution,  and  only  cut  into  pieces 
after  forty-eiglit  hours.  By  thus  removing  the  blood-corpuscles 
the  retiform  tissue  of  the  pulp  is  better  seen. 

Injected  spleen.— The  spleen  may  have  been  injected  in 
the  animal  which  was  injected  entire  ;  if  this  is  not  the  case, 
a  special  injection  is  to  be  made  from  the  splenic  artery. 
When  successfully  accomplished,  the  vessels  are  as  usual 
ligatured  to  prevent  the  escape  of  the  injection.     When  the 


240  PKACTICAL  HISTOLOGY 

gelatine  has  set  the  organ  is  cut  up  and  pieces  placed  either  in 
3  per  cent,  bichromate  of  potash  or  in  75  per  cent,  spirit.  The 
sections  will  show  what  at  first  sight  look  like  accidental 
extravasations  ;  patches,  namely,  of  injection  distributed  all 
over  the  organs,  with  the  exception  of  large  round  white 
masses,  here  and  there,  pervaded  by  a  few  capillaries.  The 
white  masses  are  sections  of  the  Malpighian  corpuscles,  and 
the  part  permeated  by  the  injection  is  of  course  the  pulp,  into 
which  the  arterial  capillaries  freely  open. 

Splenic  cells. — To  obtain  specimens  of  the  spleen  substance, 
which  will  show  in  a  separated  condition  the  cellular  elements 
which  it  contains,  and  of  which  it  is  composed,  a  small  portion 
of  the  fresh  organ  may  be  teased  out  with  needles  in  a  little 
salt  solution  or  serum.  But  it  will  be  found  that  so  much 
blood  is  incorporated  with  the  spleen  substance  (it  forms,  in 
fact,  most  of  the  soft  matter  which  can  be  expressed  from  the 
fresh  section)  that  the  view  of  the  other  parts  is  obscured  by 
innumerable  red  blood  corpuscles.  Hence  before  teasing  a 
piece  it  should  be  placed  for  forty-eight  hours  in  one-third  or 
one-fourth  alcohol.  This  decolourises  the  red  corpuscles  whilst 
preserving  the  character  of,  and  at  the  same  time  macerating 
somewhat,  the  proper  substance  of  the  spleen,  so  that  the  cells 
are  now  readily  separated  and  seen.  By  far  the  greater 
number  are  lymph  corpuscles  from  the  lymphoid  tissue  of 
the  Malpighian  nodules  and  of  the  arterial  adventitia.  But, 
besides  these,  other  cells  (splenic  cells)  are  met  with ;  larger, 
rounded  or  flattened,  and  some  of  them  containing  pigment 
granules  as  already  intimated.  They  may  be  found  either 
entirely  isolated  or  adhering  to  the  network  of  the  retiform 
tissue.  Their  nuclei,  as  well  as  those  of  the  lymphoid  cells, 
are  well  brought  out  if  a  little  hsematein  solution  is  permitted 
to  pass  under  the  cover-glass.  In  the  fresh  preparations,  not 
treated  by  bichromate  of  potash  or  any  other  reagent,  but 
made  in  serum,  some  of  the  cells  may  perhaps  be  found  con- 
taining' red  blood  corpuscles  in  their  interior,  and  transitions 
from  these  to  those  containing  pigment  are  met  with. 


THE    SUPRARENALS  241 

Reticulum.— The  reticulum  of  tlie  spleen  is  best  exhibited 
in  preparations  made  by  the  silver  chromate  method  of  Golgi. 

THE   SUPRARENAL   CAPSULES 

Sections. — To  prepare  the  suprarenal  capsule  it  is  separated 
from  the  suri-ounding  fat,  divided  into  two  or  three  pieces  by 
transverse  cuts,  and  placed  in  3  per  cent,  bichromate  of 
potash  solution  for  fourteen  days,  when  the  hardening  may  be 
completed  in  spirit  in  the  usual  manner.  Hardening  the 
organ  in  corrosive  sublimate  or  in  formol  or  in  picric  acid 
followed  by  spirit  also  gives  good  results.  The  mode  of  pre- 
paring the  sections,  which  should  include  both  cortical  and 
medullary  substance,  calls  for  no  special  description. 

Sections  of  the  injected  organ  should  also  be  prepared. 

Teased  prepa  ations. — In  a  teased-out  preparation  of  the 
fresh  organ  the  cellular  elements  of  the  cortical  and  medul- 
lary substance  may  respectively  be  studied,  and  the  effect  of 
a  solution  of  bichromate  of  potash  in  colouring  the  medullary 
cells  brown  may  be  observed. 


242  PE ACTIO AL   HISTOLOGY 


CHAPTER  XVIII 

THE    KIDNEY 

The  uninjected  kidney. — The  kidney  is  hardened  in  the 
same  way  as  the  liver  and  spleen  ;  the  yellow  chromates  of 
potash  and  of  ammonia  may  also  be  employed  (in  5  per  cent, 
solution).  The  piece  or  pieces  that  are  taken  should  include 
both  cortical  and  medullary  parts ;  but  at  the  same  time 
should  not  be  thicker  than  from  an  eighth  to  a  quarter  of 
an  inch,  otherwise  the  preservative  fluid  will  not  penetrate 
rapidly  enough  to  the  deeper  parts.  They  are  to  remain 
in  the  bichromate  solution,  if  this  is  employed,  for  two  or 
three  weeks  ;  are  then  placed  in  weak  spirit,  and  in  twenty - 
four  hours  transferred  to  strong  spirit.  In  three  or  four  days 
more  the  pieces  are  firm  enough  to  cut.  Or,  after  being 
hardened  by  the  bichromate  solution  the  pieces  may  be  simply 
soaked  in  gum  and  cut  frozen,  without  passing  through  spirit 
at  all.  Sections  as  thin  as  possible  are  to  be  made  in  a  plane 
vertical  to  the  surface  of  the  organ,  and  large  enough  to 
include  both  cortical  substance  and  Malpighian  pyramid.  One 
such  section  is  simply  mounted  in  glycerine ;  others  are 
stained,  and  after  treatment  by  the  customary  processes 
mounted  in  balsam.  These  sections  will  show — in  the  cortical 
substance  the  Malpighian  corpuscles,  the  convoluted  tubules 
variously  cut,  and  the  prolongations  of  the  straight  tubules  of 
the  medullary  substance  and  of  the  tubules  of  Henle  ;  in  the 
pyramidal  part  the  two  last-named  tubules,  and  the  collecting 
and  excretory  tubules,  seen  longitudinally,  with  a  large  number 
of  blood-vessels  running  parallel  to  and  between  them. 


THE    KIDNEY  243 

Transverse  (tangential)  sections  also  should  be  obtained 
both  from  the  cortex  and  from  the  medullary  substance  as 
well  as  sections  across  a  papilla. 

The  injected  kidney. — The  blood-vessels  of  the  kidney  will 
very  probably  be  tilled  in  injecting  an  animal  entii'e  ;  but, 
if  this  should  not  have  been  the  case,  it  is  not  difficult  to 
make  a  special  injection  of  the  separated  organ  from  the  renal 
artery.  The  red  gelatine  injection  may  be  used,  and  the 
kidney  is  kept  warm,  and  the  injection  maintained  for  a  con- 
siderable time,  in  order  that  the  vessels  of  the  glomeruli  and 
the  network  of  capillaries  in  the  cortical  substance  supplied  by 
their  efferent  vessels  may  be  completely  filled.  The  organ  is 
then  set  aside  in  a  cool  place  (surrounded  by  ice,  if  possible), 
and,  when  the  gelatine  is  completely  set,  is  cut  into  three  or  four 
pieces  and  hardened,  as  usual,  with  alcohol  or  bichromate  of 
potash.  The  sections,  which  need  not  be  very  thin,  but  should 
be  quite  even  and  comprise  the  whole  thickness  of  the  organ, 
are  to  be  mounted,  unstained,  in  balsam. 

Uriniferous  tubules.— The  uriniferous  tubules  may  be  injected 
from  the  ureters  for  a  considerable  part  of  their  length,  simul- 
taneously, if  it  be  desired,  with  the  above-described  injection  of 
the  blood-vessels,  by  a  solution  of  Berlin  blue.  But  even  if  well 
filled  they  are  too  densely  arranged  to  render  it  possible  to  trace 
individual  tubules  along  then-  whole  extent,  except  m  very  yoiing 
animals.  This  may  be  better  accomplished  b3'  niakmg  teased 
preparations  of  the  kidneys  of  small  animals,  which  have  mider- 
gone  some  process  of  preparation,  having  for  its  object  the  solu- 
tion or  softening  of  the.intertubular  substance.  Several  such  pro- 
cesses have  been  proposed,  but  none  yield  entirely  satisfactory 
residts.  The  best,  perhaps,  consists  in  digesting  tolerably  thick 
slices  of  a  small  kidney  in  a  mixtm-e  of  4  parts  of  spirit  and  1  of 
hydrocliloric  acid,  kept  boUmg  for  3  or  4  hours.  The  boiling  is 
performed  in  a  flask  fitted  with  a  condenser,  in  which  the  vapour 
which  is  driven  off  by  the  boiling  becomes  condensed  and  flows 
down  again  into  the  flask.  The  slices  are  then  placed  in  \\ater, 
and,  after  lying  in  this  for  a  few  days,  minute  slureds,  comprising 
the  whole  depth  from  external  surface  to  pai)ilhe,  are  split  ofi"  with 
needles,  placed  on  a  slide,  and  unravelled  as  much  as  possible  by 

li  2 


244  PKACTICAL  HISTOLOGY 

aid  of  the  dissecting  microscope.  The  preparation  is  covered  with 
a  specially  large  piece  of  covering-glass  (a  hair  being  first  added  to 
avert  the  pressure  of  the  glass  on  the  soft  tubules),  and  stained  by 
drawing  picric  acid  solution  under  the  cover-glass.  This  soon 
colours  the  tissue  intensely  yellow  ;  glycerine  may  then  be  allowed 
to  pass  in  at  the  border  in  order  to  complete  the  preparation. 
Some  of  the  tubules  will  be  found  isolated  for  a  considerable  part 
of  their  length,  and  the  passage  of  the  convoluted  tubules  into  the 
looped  tubes  of  Henle  may  especially  be  well  seen.  The  epithelium 
of  the  tubules  is  for  the  most  part  well  preserved. 

Examination  of  the  fresh  kidney. — When  by  these  various 
means  sufficient  acquaintance  has  been  gained  with  the  various 
tubules  and  their  contents  in  situ,  the  examination  of  the 
fresh  tissue  in  serum  may  be  attempted.  With  this  object 
small  snips  are  to  be  made  from  different  parts  of  a  freshly- 
cut  surface  with  a  pair  of  curved  scissors  and  teased  out  in 
a  drop  of  serum,  with  the  aid  of  the  dissecting  microscope, 
so  as  to  separate  as  many  of  the  tubules  as  possible.  In  doing 
this  much  of  the  epithelium  will  become  detached,  and  the 
characters  of  the  individual  cells  in  the  fresh  condition  may  be 
studied. 

The  outlines  of  the  cells  of  the  capsules  of  Bowman  and  of  the 
tubules  may  be  shown  by  nitrate  of  silver.  For  this  pm-pose  a 
fresh  kidney  is  sliced  in  half  by  a  single  cut  with  a  sharp  razor 
in  the  direction  of  the  tubules.  One  of  the  halves  is  thoroughly 
washed  with  distilled  water,  and  solution  of  nitrate  of  silver 
(5-  per  cent.)  is  poured  over  the  cut  surface.  After  a  minute  and 
a  half  the  silver  solution  is  rinsed  off  with  distilled  water,  and 
the  piece  of  kidney  is  placed  in  water,  with  the  silvered  surface 
exposed  to  the  sunlight.  When  brown  it  may  be  removed  from  the 
light,  and  placed  in  spirit  for  twenty-four  hours ;  one  or  two 
sections  are  then  made  from  the  brown  surface,  clarified  in  oil  of 
cloves,  and  mounted  in  balsam. 

THE    URETERS 

Sections. — The  ureters  are  prepared  in  the  same  way  as 
the   intestine — by  moderately  distending  an  excised   portion 


THE    URINARY    liLADDER  245 

with  some  sort  of  hardening  solution,  and  placing  the  piece 
thus  distended  in  a  beaker  containing  some  of  the  same 
mixture.  After  a  few  hours  the  tube  is  slit  open,  and  trans- 
ferred to  spirit  after  two  or  three  days.  The  sections  arc  to 
be  made  across  the  length  of  the  tube,  and  stained  and 
mounted  in  the  ordinary  manner. 

Epithelium  of  ureter.  — To  study  the  separated  epithelial 
cells  a  piece  as  fresh  as  possible  is  cut  open,  pinned  out  on  a 
cork  with  the  inner  surface  uppermost,  and  immersed  in  one- 
third  alcohol  or  ^-  per  cent,  bichromate  of  potash  solution  for 
from  twenty-four  to  forty-eight  hours.  Some  of  the  epithe- 
lium is  then  scraped  off  with  a  spear-shaped  needle  or  the  end 
of  a  scalpel,  and  is  broken  up  in  a  drop  of  water.  After  the 
addition  of  a  piece  of  hair  to  the  fluid  the  cover-glass  may  be 
applied,  and  the  preparation  examined  with  a  high  power. 
If  it  prove  successful,  with  many  of  the  epithelial  cells  fully 
separated,  it  may  he  permanently  preserved.  With  this 
object  the  cells  should  6rst  be  stained,  by  allowing  weak 
hremalum  solution  to  run  under  the  edge  of  the  cover- 
glass.  The  h?emalum  is  to  be  followed  by  a  drop  of  glyce- 
rine applied  at  the  same  edge ;  and,  when  the  glycerine 
has  become  diffused  underneath,  all  that  is  necessary  is  to 
cement  the  cover-glass. 

THE    BLADDER 

The  urinary  bladder,  both  for  sections  and  teased-out 
preparations,  is  prepared  by  exactly  the  same  methods  as  the 
ureters.  To  distend  it  a  glass  cannula,  connected  by  an 
indiarubber  tube  with  a  bottle  containing  the  hardening  fluid, 
is  tied  into  the  urethra.  The  organ  must  not  be  o\er-dis- 
tended,  but  only  moderately  filled.  Any  urine  which  it  may 
contain  should  first  be  allowed  to  run  out  through  the 
cannula. 

In  dealing  with  the  human  bladder  or  the  bladders  of 
larsre  animals  it  is    more   convenient  to  cut  out  one  or  two 


246  PEACTICAL  HISTOLOGY 

pieces  from  different  parts  and  to  pin  them  on  a  cork  or  cake 
of  wax  for  immei^sion  in  the  hardening  fluid. 

The  musculature  of  the  bladder  in  the  frog  has  already 
been  studied  (p.  135).  Such  a  preparation  can  also  be  made 
of  the  bladder  of  a  small  mammal,  and  will  serve  to  show  the 
lymphatics  as  well.  The  nerves  may  be  shown  by  any  of  the 
special  methods  detailed  in  the  chapter  on  nervous  tissue. 


247 


CHAPTER  XIX 

THE    GENERATIVE   ORGANS 

Erectile  tissue. — Those  parts  which  contain  erectile  tissue 
will  be  best  studied  after  having  been  injected.  Their  blood- 
vessels and  sinuses  may  have  been  filled  in  the  animal  which 
was  injected  entire  from  the  root  of  the  aorta  ;  but  if  not, 
a  special  injection  from  the  lower  end  of  the  abdominal  aorta 
is  to  be  made,  the  arteries  supplying  the  lower  limbs  being 
first  tied  to  prevent  waste  of  the  injection.  The  hardening 
of  the  parts  in  spirit  nmst  be  effected  very  gradually  (the 
spirit  being  daily  made  stronger),  since  otherwise  the  gelatine 
shi'inks  away  from  the  walls  of  the  venous  sinuses,  and  the 
preparation  becomes  in  great  measui'e  spoiled.  This  is,  how- 
ever, obviated  by  using  bichromate  of  potash.  The  sections 
should  some  of  them  be  mounted,  unstained,  in  balsam,  others 
after  being  lightly  stained  with  hsematein,  so  that  the  plain 
muscular  and  fibrous  tissue,  and  also,  in  sections  including  the 
urethra,  the  epithelium  of  that  tube  may  be  exhibited  as  well 
as  the  vessels. 

Parts  which  have  not  been  injected  ai'e  hardened  in  3  per 
cent,  bichromate  of  potash  solution,  0-2  per  cent,  chromic  acid 
or  saturated  solution  of  picric  acid,  followed  by  spirit  in  either 
case. 

Prostate  and  seminal  vesicles. — The  glandular  organs, 
such  as  the  prostate  and  vesiculre  seminales,  are  prepared  in 
the  same  way  or  with  spirit  only. 

Scrotum,  labia,  &c. — The  scrotum,  and  labia,  and  the 
vagina  are  prepared  in  the  same  way  as  the  skin  (see  p.  199). 


248  PEACTICAL  HISTOLOGY, 

Uterus. — The  human  uterus  is  best  hardened  in  3  per 
cent,  bichromate  of  potash  ;  its  cavity  should  be  freely  laid 
open.  Sections  may  be  cut  from  it  by  the  freezing  method. 
In  animals  (the  rabbit,  for  instance),  where  it  is  more  mem- 
branous, the  uterus  and  the  upper  part  of  the  vagina  may 
be  prepared  together  by  distending  them  with  picric  or 
chromic  acid  solution  through  a  cannula  tied  into  the  lower 
part  of  the  vagina.  The  vagina  is  then  tied  uj),  and  the 
organs  are  cut  out  and  placed  in  a  quantity  of  the  solution  ; 
in  three  or  four  hours  they  are  laid  open  and  the  fluid  renewed, 
and  in  a  day  or  two  are  ready  to  be  put  into  spirit.  The 
sections  are  stained  and  mounted  in  the  usual  way. 

Section  of  ovary. — The  ovaries  are  prepared  by  placing 
them — with  as  little  handling  as  possible,  so  as  to  avoid  rub- 
bing off  the  columnar  epithelium  which  covers  the  surface — in 
0"2  per  cent,  chromic  acid  or  in  picric  acid  or  in  formol  (whole  if 
taken  from  a  small  animal,  such  as  a  rabbit  or  cat ;  cut  into  two 
or  three  pieces  if  from  a  larger  one).  In  most  of  the  lower 
animals  they  must  be  sought  much  higher  in  the  abdomen  than 
in  the  human  female  ;  in  the  rabbit  they  occur  as  small  elon- 
gated bodies,  dotted  all  over  with  little  projections  (the  Graa- 
fian follicles),  and  situated  just  below  the  kidneys.  They  are 
left  in  the  chromic  solution  for  seven  days,  and  then  placed  in 
spirit,  which  must  be  changed  frequently,  and  in  a  few  days 
more  are  ready  for  cutting.  The  sections  are  to  be  stained 
in  hsemalum,  carmalum,  or  picro-carmine  solution  ;  hsemalum 
sometimes  colours  very  deeply  the  coagulated  fluid  in  the 
Graafian  follicles,  so  that  the  epithelial  contents  are  obscured. 

The  ovum. — The  ripe  mammalian  ovum,  although  it  can 
be  seen  within  the  larger  Graafian  follicles  in  the  sections  of 
the  hardened  organ,  forms  a  much  more  beautiful  object 
when  obtained  isolated  from  the  ovary  of  a  recently-killed 
animal.  A  full-grown  doe  rabbit,  not  pregnant,  is  to  be 
sacrificed  for  this  purpose.  One  of  the  ovaries  having  been 
removed,  it  is  held  firmly  between  the  finger  and  thumb  over 
a  clean  glass  slide,  in  such  a  position  that  the  largest  and 


THE   TESTICLH  249 

most  prominent  Graafian  follicle  is  almost  in  contact  with 
the  middle  of  the  slide.  The  follicle  is  then  pricked  with  a 
sharp-pointed  scalpel,  so  as  to  cause  the  fluid  contents  of  the 
follicle  to  spirt  out,  carrying  with  them  the  ovum,  surrounded 
by  a  number  of  the  epithelium  cells.  The  ovum  is  i-ather  too 
small  to  be  detected  with  the  naked  eye,  but  its  pi-esence  may 
be  suspected  if,  on  glancing  at  the  slide  in  such  a  manner  that 
the  light  is  reflected  from  the  surface  of  the  fluid  to  the  eye, 
a  slight  prominence  is  observed  on  the  otherwise  flat  surface. 
Its  presence  here  is  confirmed  by  examination  with  a  low 
power,  and  it  may  then  be  carefully  obsei-ved  with  the  ordinary 
high  power.  It  is  better,  if  possible,  not  to  apply  a  cover- 
glass,  for  the  zona  pellucida  is  apt  to  become  broken  ;  and, 
moreover,  even  slight  pressure  spoils  in  great  measure  the 
natural  appearance  of  the  object.  But  if  the  objective 
l)eeomes  dimmed  by  its  proximity  to  the  fluid,  or  if  it  is 
desired  to  employ  an  immersion,  then  a  thin  cover-gla3S  must 
be  used,  and  to  protect  the  ovum  from  pressure  a  narrow  slip 
of  thick  paper  (an  ordinary  hair  is  not  thick  enough)  is  to  be 
put  on  either  side  before  the  cqver-glass  is  applied.  If  the 
fluid  which  accompanies  the  o^'um  from  the  Graafian  follicle 
is  not  in  sufficient  quantity,  a  drop  of  aqueous  humour  may 
be  added  to  it.  It  is  not  possible  to  preserve  this  preparation 
very  satisfactorily. 

Section  of  uninjected  testis. — The  testis  is  best  hardened 
in  formol  or  absolute  alcohol.  If  from  a  large  animal  it  should 
be  cut  into  in  two  or  three  places  before  being  plunged  in 
the  fluid.  It  varies  in  consistence,  being  firmest  in  those 
animals  (cat,  pig)  in  which  the  peculiar  granular  polyhedral 
cells,  which  accompany  and  surround  the  blood-vessels,  are 
most  numerous.  Thin  pieces  may  be  stained  in  bulk  with 
dilute  hajmalum  or  carmalum.  Sections  of  both  the  body  of 
the  testis  and  the  epididymis  are  to  be  made. 

Lymphatics  of  testis. — In  the  sections  made  as  above  there 
will  be  observed  in  the  interstices  between  the  seminiferous 
tubules  large  cleft-like  spaces,  looking  almost  like  accidental 


250  PEACTICAL  HISTOLOGY 

clefts  in  the  loose  connective  tissue  uniting  the  tubules.  They 
are  in  reality,  however,  the  lacunar  commencements  of  the 
lymphatics.  To  show  this  the  following  simple  experiment 
may  be  performed  : — In  a  recently-killed  dog  the  fine  cannula 
of  a  hypodermic  syringe,  tilled  with  Berlin  blue  solution,  is 
stuck  through  the  scrotum  into  the  middle  of  the  substance 
of  one  of  the  testes,  and  the  solution  is  slowly,  and  without 
exerting  any  considerable  pressure,  injected  into  the  organ. 
If  the  abdomen  is  opened  the  blue  fluid  will  soon  be  seen 
passing  along  the  lymphatics  which  run  in  the  spermatic  cord, 
and  from  these  into  those  of  the  back  of  the  pelvis  and 
abdomen,  at  length  reaching  the  thoracic  duct.  If  now  the 
testis  is  removed  and  hardened  in  spirit,  and  sections  are 
made  of  the  hardened  organ,  it  will  be  found  that  the  inter- 
tubular  spaces  are  occupied  by  the  blue  substance,  and,  since 
they  are  proved  by  the  injection  to  be  in  free  communication 
with  the  lymphatics  which  leave  the  organ,  the  spaces  are 
to  be  looked  upon  as  giving  origin  to  the  lymphatics. 

Isolation  of  the  seminiferous  tubules  and  elements. — 
For  obtaining  the  tubules  isolated  for  a  considerable  length, 
pieces  of  the  testis  (preferably  human)  are  placed  for  a  day  or 
two  in  hydrochloric  acid,  diluted  with  one-third  its  volume 
of  water,  and  maintained  at  30°  C.  They  are  then  allowed  to 
lie  in  water  until  the  tubules  can  be  readily  separated  with 
needles.  Teased-out  preparations  of  fragments  of  testicle 
which  have  been  macerated  in  one-third  alcohol  for  two  or 
three  days  may  also  be  made.  Fragments  of  the  fresh 
testis-substance  should  also  be  teased  in  saline,  to  exhibit 
the  form,  stages  of  development,  and  movements  of  the 
spermatozoa.  For  the  object  last  named  the  preparation 
should  be  examined  on  the  warm  stage. 

Epithelioid  cells  of  seminiferous  tubules. — To  exhibit  the 
fact  that  the  apparently  structureless  basement  membrane  of 
the  seminiferous  tubules  is  in  reality  composed  of  layers  of 
flattened  epithelioid  cells,  a  portion  of  the  testicular  substance 
is   partially  unravelled  in  distilled  water,   and   some  of  the 


THE   MAMMAIIY   GLANDS  251 

tubules  which  are  thus  isolated  are  dipped  into  nitrate  of 
silver  solution  f(»r  a  minute,  and,  after  being  again  rinsed  in 
water,  are  mounted  in  glycerine  and  exposed  to  the  light  ; 
the  lines  of  junction  between  the  flattened  cells  are  by  this 
means  made  evident. 

Tunica  vaginalis. — The  tunica  vaginalis  is  to  be  prepared 
in  the  same  way  as  the  other  serous  membranes  (with  nitrate 
of  silver),  partly  unbrushed  to  show  the  epithelioid  covering, 
and  partly  brushed  for  the  sake  of  exhibiting  the  parts 
beneath.  For  the  preparation  of  the  visceral  part,  the  pro- 
cess is  similar  to  that  adopted  for  the  pericardium  covering 
the  surface  of  the  heart  (p.  205),  and  need  not  here  be  more 
particularly  described. 

The  mammary  glands. — Small  pieces  of  these  organs  are 
hardened  by  being  placed  in  0'2  chromic  acid  solution  for  a 
week  or  ten  days,  or  in  picric  acid  for  a  day  or  two,  or  in 
forniol  or  corrosive  sublimate,  subsequently  transferring 
them  to  spirit.  Absolute  alcohol  may  also  be  used  at  once, 
and  produces  the  desired  result  more  rapidly  ;  the  sections 
stain  more  readily  than  if  chromic  or  picric  acid  is  used. 
The  appearances  presented  by  the  cells  of  the  alveoli  vary 
considerably  according  to  the  state  of  functional  activity  of 
the  gland.  It  will  also  be  found  that  the  human  mammary 
gland  is  much  less  compact,  its  glandular  substance  l^eing 
more  scattered  in  the  connective  tissue  of  the  organ  than  is 
the  case  in  most  animals. 


252  PEA  OTIC  AL  HISTOLOGY 


CHAPTER  XX 

THE  CENTRAL  NERVOUS  SYSTEM  ;  THE  BRAIN  AND 
SPINAL  CORD 

Preparation  by  silver  chromate. — Some  experience  has 
already  been  obtained  of  the  raethods  which  are  employed  for 
studying  the  cellular  elements  of  the  central  nervous  system 
(pp.  150-153).  The  silver  chromate  method  of  Golgi  is  to  be 
employed  as  there  directed  for  studying  portions  of  the  cerebral 
cortex,  of  the  cerebellar  cortex,  of  the  medulla  oblongata,  and  of 
the  spinal  cord.  In  all  cases  it  will  be  of  advantage  to  employ 
f cetal  or  young  animals,  but  this  is  not  necessary,  for  veiy  good 
preparations,  especially  of  cerebral  cortex,  are  yielded  by  adult 
organs  treated  by  this  method.  The  spinal  cord  was  the  part 
then  under  investigation,  but  the  nerve- cells  found  in  the  grey 
matter  of  the  cerebrum  and  of  the  cerebellum  may  be  observed 
in  the  same  manner  with  equally  satisfactory  results.  With- 
out delaying,  then,  to  repeat  the  directions  there  given,  we 
may  pass  on  to  describe  the  best  general  methods  of  preparing 
sections  of  the  parts  in  question. 

Preparation  with  bichromate  of  ammonia. — To  harden 
any  part  of  the  central  nervous  system  for  histological 
purposes  the  most  generally  useful  reagents  are  the  bichromates 
of  potash  and  of  ammonia  (2  or  3  per  cent,  solution).  These 
will  in  three  or  four  weeks  render  the  tissue  sufficiently  firm 
for  obtaining  thin  sections,  which  may  be  made,  after  a 
preliminary  soaking  in  gum,  by  the  freezing  method.  For 
celloidin-embedding  it  is  necessary  to  complete  the  hardening 


WKIGERT-PAL   .MKTllOI)  253 

first  witli  weak  and  then  with  strong  spirit.  The  pieces  to  be 
hardened  should  not  be  too  large,  oi-  at  least  not  too  thick, 
but  the  bichromate  solution  has  considerable  powei-  of  pene- 
tration, and  the  whole  length  of  the  spinal  cord  of  any  of 
the  smaller  quadrupeds,  and  even  that  of  man,  may  be 
hiirdened  in  it  intact  if  put  in  perfectly  fresh.  It  is  always 
])etter,  however,  to  cut  it  into  short  lengths.  Ten  per  cent,  of 
formol  solution  has  of  late  come  into  use  in  place  of  bichromate 
of  potash.  It  penetrates  even  more  readily,  and  hardens 
much  more  rapidly.  To  stain  by  Nissl's  method  (see  next 
page)  alcohol  only  may  be  employed. 

In  this  way  a  piece  of  the  cerebellum,  two  or  three  small 
pieces  from  different  parts  of  the  convoluted  surface  of  the 
cerebrum,  the  medulla  oblongata,  and  pieces  of  the  spinal  cord 
from  the  middle  of  the  three  regions  (cervical,  dorsal,  and 
lumbar)  are  to  be  prepared. 

Preparation  of  sections  of  the  central  nervous  system. — 
Although  it  is  possible  to  cut  small  pieces  of  the  central 
nervous  system  sufficiently  thin  either  by  the  freezing  method 
or  by  an  inclined  plane  microtome,  with  the  knife  wetted  with 
spirit,  for  larger  sections  and  when  it  is  very  necessary 
to  keep  the  neighbouring  parts  (nerve-roots  and  the  like) 
in  position,  the  collodion  method  must  be  used  (see  p.  29). 
For  staining  by  Nissl's  method,  it  is  desirable  to  imbed  in 
parafl&n. 

Staining  of  the  sections.  Weigert-Pal  method. — Sections 
which  have  been  prepared  by  the  liichromate  method  may  be 
stained  in  the  ordinary  way  with  carminate  of  soda  (-5  p.c),  car- 
malum,  or  aniline  blue-black.  Acid  fuschin  has  also  been  re- 
commended by  Weigert  as  a  reliable  stain.  A  saturated  water 
solution  is  used,  and  the  sections  are  partly  decolourised  by 
absolute  alcohol  rendered  very  faintly  alkaline  by  caustic 
potash,  and  are  then  washed  with  water  and  alcohol, 
and  mounted  in  Canada  balsam.  But  for  exhibiting  the 
medullated  hbres,  and  the  tracts  which  they  form,  the  follow- 
ing modification  of  a  method  originally  devised  by  Weigei-t, 


254  PEACTICAL  HISTOLOGY 

and  known  by  his  name,  will  be  found  the  best  : — The 
sections,  whether  cut  by  the  freezing  method  or  by  the 
collodion  process,  are  placed  first  in  water  for  a  few  minutes  ; 
then  for  a  few  hours  in  Marchi's  solution,  which  is  a  mixture 
of  Miiller's  fluid  (2  parts)  with  1  per  cent,  osmic  acid  (1 
part).  They  are  then  washed  in  water  and  transferred  to  acid 
hsematoxylin  (p.  19),  in  which  they  are  left  overnight;  by 
which  time  they  are  completely  black.  They  are  next 
thoroughly  washed  with  tap  water  and  put  first  into  0-25  per 
cent,  solution  of  permanganate  of  potash  for  five  minutes, 
then  rinsed  in  water  and  transferred  to  Pal's  solution  (sodic 
sulphite  1  g.  ;  oxalic  acid  1  g.  ;  distilled  water  200  c.c). 
This  bleaches  the  grey  matter,  leaving  the  medullary  sheath  of 
all  nerve-fibres  black.  The  process  of  differentiation  usually 
takes  but  a  few  minutes,  but  if  they  are  not  properly  differen- 
tiated in  half  an  hour  they  are  replaced,  after  washing,  in  the 
permanganate  solution  for  a  few  minutes  and  then  again  placed 
in  Pal's  solution.  Finally  they  are  well  washed  with  tap 
water,  and  transferred  through  alcohol  of  increasing  strength 
and  oil  of  bergamot  into  xylol  balsam.  This  modification 
has  many  advantages  over  the  original  methods  of  Weigert 
and  Pal. 

The  sections  may  before  mounting  be  stained  with  eosin  to 
show  the  nerve-cells. 

Nissl's  method. — The  tissue  is  hardened  in  alcohol  (96  p.c. 
or  absolute),  which  is  changed  after  the  first  day.  It  is  left 
in  alcohol  from  two  to  six  days,  then  simply  embedded,  not 
soaked,  in  paraffin.  The  sections  are  stained,  after  removing 
the  paraffin,  by  immersion  for  a  few  minutes  in  a  0*5  p.c. 
aqueous  solution  of  methylene  blue,  which  has  been  just  pre- 
viously heated  to  about  70°  C.  for  half  to  one  minute.  They 
are  then  differentiated  by  alcohol  or  aniline-oil  alcohol  (1  to  5), 
passed  through  origanum  oil,  and  mounted  in  xylol  balsam. 
The  nerve-cells  and  their  processes  are  beautifully  stained,  the 
methyene  blue  being  retained  especially  by  the  cell-granules. 
A  double  staining  may  be  got  by  using  alcoholic  solution  of 


NERVOUS  SYSTEM  255 

eosiri  of  orange  G,  or  of  acid  fuschiii,  as  a  differentiating 
reagent. 

Weigert's  method  for  showing  neuroglia-cells. — After 
hardening  in  2  per  cent,  bichromate  of  potash,  followed  by 
alcohol,  sections  are  cut  by  the  celloidin  method,  and  tirst 
stained  slightly  with  carmine.  Tliey  are  next  stained  with 
gentian-violet  as  follows  : — A  water  solution  of  the  dye  is  made 
by  adding  a  few  drops  of  a  saturated  alcohol  solution  to 
aniline  water  (see  p.  21).  After  staining,  the  sections  are 
decolourised  in  iodine  solution  (1  per  cent,  dissolved  in  2  per 
cent,  solution  of  potassic  iodide).  After  two  or  three  minutes 
they  are  transferred  to  a  mixture  of  aniline  oil  (2  pai'ts)  and 
xylol  (1  part).  When  sufficiently  decolourised  they  are  well 
washed  with  xylol  and  mounted  in  xylol  balsam. 

Tracts  of  conduction  in  the  central  nervous  system. — To 
study  the  tracts  of  conduction  in  the  central  nervous  system 
two  methods  may  be  employed.  One  is  to  prepare  by  the 
Weigert-Pal  process  sections  from  the  several  parts  in  the 
foitus  and  new-born  infant.  Those  tracts  in  which  the 
medullary  sheath  of  the  nerve -fibres  is  still  incomplete  appear 
in  such  sections  unstained,  whereas  those  in  which  it  is  com- 
plete are  stained  black  :  in  this  way  many  of  the  principal 
tracts  of  conduction  {e.g.  the  pyramidal,  which  develop  late) 
are  differentiated  from  the  rest  of  the  white  matter.  The 
other  method  consists  in  producing  degenerations  of  certain 
tracts  and  staining  the  degenerated  nerve  tibres.  By  the 
Wallerian  law  of  degeneration,  any  nerve-fibre  which  is 
separated  from  its  nerve- cell  undergoes  a  process  of  degenera- 
tion which  is  characterised  by  its  medullary  sheath  breaking 
up  into  droplets  of  myelin.  These  droplets  of  myelin  are  much 
more  susceptible  to  osmic  stain  than  normal  medullary  sub- 
stance, and  advantage  is  taken  of  this  fact  in  the  method 
employed  to  show  the  tracts. 

The  first  thing  is  to  produce  the  required  lesion,  such  as  a 
complete  transverse  section  or  a  hemisection  of  the  spinal 
cord,  a  removal  of  a  portion  of  cerebral  cortex,  and  the  like. 


256  PEACTICAL  HISTOLOGY 

The  operation  is  done  aseptically  and  the  external  wound 
should  heal  at  once.  The  animal  is  kept  alive  for  two  or  three 
weeks.  It  is  then  killed,  and  the  organs  to  be  investigated 
are  placed  in  2  per  cent,  bichromate  solution  or  Miiller's 
fluid  for  a  few  days ;  thinnish  slices  are  then  cut  from 
different  parts  and  laid  on  cotton-wool  in  a  considerable 
quantity  of  Marchi's  solution  (each  one  in  a  separate  vessel), 
which  should  be  changed  in  a  day  or  two  if  it  lose  the  odour 
of  osmic  acid.  They  are  left  in  the  Marchi  fluid  for  a  week, 
and  are  then  washed  with  water  and  placed  in  spirit.  Sections 
are  cut  by  the  collodion  method,  and  are  not  further  stained, 
but  are  simply  passed  through  alcohol  and  cedarwood 
oil  to  be  mounted  in  balsam.  The  degenerated  tracts  stand 
out,  even  with  the  naked  eye,  as  black  patches  upon  the 
generally  colourless  ground  of  the  section. 

It  has  not  hitherto  been  possible  to  produce  this  Marchi  staining 
of  degenerated  nerve-fibres  in  individual  sections,  but  only  in  thin 
pieces  of  the  tissue.  Hamilton  has  devised  a  method  whereby  the 
staining  can  be  effected  in  the  sections.  Instead  of  taking  fresh 
bichromate  of  potash,  he  uses  bichromate  solution  which  has  been 
already  used  for  hardening  pieces  of  brain,  adds  1  c.c.  of  1  per  cent, 
osmic  acid  to  200  c.c.  of  this  solution,  and  keeps  the  sections  twenty- 
four  hours  in  the  mixture  at  about  38°  C.  They  are  then  washed 
and  transferred  for  a  short  time  to  the  following  solution  : 

Pyrogallic  acid         ....  1  grm, 

75  per  cent,  alcohol          ...  50  cc. 

Glycerine 50  cc. 

Distilled  water          ....  300  cc. 

then  rmsed  in  water,  placed  for  3  days  in  0'25  per  cent,  permanga- 
nate of  potash,  washed  with  solution  of  sulphurous  acid,  and 
mounted  in  balsam  in  the  usual  way. 


257 


CHAPTER   XXI 

THE    ORGANS    OF    THE    SENSES — THE    EYE 

The  study  of  the  eye  should  be  made  as  much  as  possible  from 
that  of  the  human  subject,  for  there  are  slight  differences  in 
the  structure  of  some  of  the  parts  in  man  and  animals,  and, 
moreover,  it  is  on  the  whole  easier  to  demonstrate  the  structures 
in  the  human  eye.  On  the  other  hand,  there  is  no  organ  which 
it  is  so  absolutely  essential  to  obtain  in  a  perfectly  fresh 
condition.  For  this  and  other  reasons  it  is  scarcely  possible 
to  get  material  from  the  post-moi'tem  room,  and  the  only 
opportunities  that  usually  present  themselves  occur  when  an 
eye  is  removed  on  account  of  some  injury  or  disease  which 
is  confined  to  a  particular  part ;  the  other  intact  portions 
may  in  such  cases  be  available  for  histological  purposes.  In 
rare  instances  an  entire  healthy  eye  has  to  be  removed,  and 
the  following  would  be  perhaps  the  best  way  to  deal  with  the 
excised  oi'gan  with  the  view  of  making  the  best  use  of  it  : — As 
soon  after  removal  as  possible  separate  the  eye,  by  an  oblique 
cut  with  a  very  sharp  knife  or  razor,  into  two  halves,  an 
anterior  and  a  posterior  ;  the  cut  to  start  from  just  behind  the 
attachment  of  the  iris  anteriorly  and  superiorly,  and  pass 
downwards  and  backwards  towards  the  posterior  part  of  the 
organ,  coming  out  a  little  below  the  yellow  spot  and  optic 
nerve.  Then  put  the  posterior  part,  after  allowing  the 
vitreous  humour  to  fall  away  from  the  retina,  into  a  mixture  of 
three  parts  of  Midler's  fluid  with  1  part  of  1  per  cent,  solution 
of  osmic  acid  ;  the  anterior  part  may  be  put  into  JNIiiller's  fluid 
alone.     The  cornea  is  to  be  cut  through  at  one  place  with  a 

s 


258  PEACTICAL  HISTOLOGY 

sharp  scalpel,  so  that  the  preservative  fluid  may  get  freely  into 
the  anterior  chamber. 

The  piece  in  Mliller  and  osmic  is  left  there  for  two  to  five 
days  or  more  :  each  day  a  small  fragment  of  retina  is  to  be 
removed  and  prepared  with  silver  nitrate  by  the  method  of 
Golgi  (p.  152).  What  remains  of  the  preparation  is  after  five 
days  placed'  in  water  for  two  hours,  and  finally  transferred  to 
a  mixture  of  equal  parts  of  glycerine,  alcohol,  and  water  ;  in 
this  it  is  to  remain  for  a  week  or  more  until  wanted. 

The  other  piece  is  to  lie  in  Miiller's  fluid  for  three  weeks, 
changing  the  fluid  once  or  twice  ;  it  is  then  placed  in  weak 
spirit  for  a  day  or  two,  and  finally  preserved  in  strong  spirit. 
Eormol  (10  per  cent.)  may  be  used  in  place  of  Miiller's  fluid  ;  it 
will  be  found  much  more  rapid  in  its  action.  Of  the  lower 
animals,  the  eyes  of  the  pig  serve  best  for  exhibiting  the 
minute  structure,  especially  of  the  retina.  In  this  animal  the 
eye  corresponds  more  closely  in  point  of  size  and  approaches 
more  nearly  in  structure  to  the  human  eye  than  that  of  the 
ox  or  sheep,  the  other  animals  the  eyes  of  which  are  usually 
readily  procurable. 

The  eyelids. — These  are  studied  by  making  sections  of  the 
hardened  lid  across  its  long  axis  and  vertically  to  its  surfaces. 
The  lid  may  be  obtained  from  a  still-born  child,  preferably  one 
the  blood-vessels  of  which  have  been  injected.  It  is  to  be 
hardened  in  spirit  and  embedded,  and  the  sections — which 
present  no  unusual  difficulty — stained  and  mounted  in  the  usual 
.way.  In  this  way  almost  all  the  parts  are  well  displayed  : 
the  skin  with  its  epidermis  on  the  outer  side,  and  with  a  few 
small  hairs  and  sweat-glands  seen  here  and  there  ;  the  mucous 
membrane  (conjunctiva)  on  the  inner  side  ;  the  Meibomian 
glands  cut  along  the  length  of  their  wide,  straight  duct,  with 
their  round  saccules  lined  with  epithelium  cells  (of  a  whitish 
glistening  appearance,  due  to  the  fatty  secretion  they 
contain,  and  which  also  fills  the  duct)  ;  the  eyelashes  with 
their  large  hair-follicles  and  sebaceous  glands  ;  the  cut  ends 
of  the  very  small  jnuscular  fibres  of  the  orbicularis  arranged 


THE  EYE  259 

in  groups,  separated  by  connective  tissue  ;  and  the  general 
connective  tissue  which  serves  to  unite  all  the  parts  together 
and,  becoming  denser  towards  the  inner  surface,  forms  the  so- 
called  '  tarsal  cartilage,'  long  described  as  composed  of  fibro- 
cartilage,  in  reality  containing  no  cartilage-cells. 

The  lachrymal  gland  is  prepared  in  the  same  way  as  the 
salivary  glands. 

The  suhstance  of  the  sclerotic. — The  sclerotic  is  studied 
by  means  of  sections  made  from  an  eye  that  has  been  hardened 
in  3  per  cent,  bichromate  of  potash.  They  are  made  frozen, 
after  the  tissue  has  been  soaked  in  gum.  Covering  the 
outer  surface  of  the  globe  is  a  loose  connective  tissue 
membrane,  the  capsule  of  Tenon,  composed  of  two  apposed 
layers,  lined  by  epithelioid  cells,  which  bound  a  lymph-space, 
and  covering  the  inner  surface  of  the  sclerotic  is  another 
delicate  lamella  of  loose  connective  tissue,  closely  adherent  to 
the  fibrous  substance  of  the  coat,  and  of  a  brown  appearance, 
due  to  the  presence  of  pigment.  This  layer  (the  lamina  fusca) 
is  also  bounded  internally  by  a  layer  of  epithelioid  cells,  and  is 
separated  from  a  similar  lamella  on  the  outer  surface  of  the 
choroid  coat  (the  lamina  suprachoroidea)  by  another  lymph- 
space,  traversed  here  and  there  by  the  vessels  and  nerves  as 
they  pass  obliquely  across  it  from  the  sclerotic  to  the  choroid. 

Capsule  of  Tenon. — To  exhibit  the  epithelioid  cells  of  the 
capsule  of  Tenon  a  fresh  eye  is  taken,  and  the  adhering  orbital 
fat,  and  eveiything  but  the  insertions  of  the  eye-muscles, 
removed  from  the  globe,  except  the  above-mentioned  loose 
membrane.  The  eye  thus  cleared  is  rinsed  in  distilled  water 
and  a  few  drops  of  nitrate  of  silver  solution  are  poured  over 
the  posterior  part.  After  three  minutes  the  silver  is  rinsed 
off  again  by  a  stream  of  distilled  water,  and  the  eye  is  placed 
in  water  in  the  sunlight.  When  sufficiently  stained  it  is 
removed  from  the  window,  fastened  under  water  to  a  loaded 
cork  by  a  long  pin  passed  through  the  cornea,  and  a  piece  of 
the  capsule  of  Tenon  is  dissected  off  the  globe,  floated  flat  on 
to  a  slide,  and  removed  from  the  fluid.     After  the  excess  of 


260  PEACTICAL  HISTOLOGY 

water  has  been  got  rid  of,  the  piece  is  covered  in  glycerine 
and  examined  for  the  epithelioid  markings.  Or  the  prepara- 
tion may  be  made  by  hardening  in  spirit  and  preparing 
tangential  sections  of  the  silvered  surface. 

Lamina  fusca. — The  epithelioid  layer  lining  the  lamina 
fusca  is  also  prepared  by  nitrate  of  silver.  A  piece  of  the 
sclerotic  is  dissected  off  from  a  fresh  eye  ;  the  convex  outer 
surface  of  the  piece  is  then  pressed  in  and  made  concave,  the 
previously  concave  inner  surface  being  made  the  convex  one, 
and  the  piece  is  first  dipped  in  distilled  water,  then  placed  for 
two  minutes  in  silver  solution,  then,  after  being  again  rinsed 
in  water,  transferred  to  spirit  and  placed  in  the  light,  with 
the  inner  surface  or  lamina  fusca  uppermost.  After  a  few 
minutes,  by  which  time  in  bright  diffused  daylight  the 
silver  will  probably  be  reduced,  although  owing  to  the  natural 
brown  colour  this  cannot  well  be  seen,  it  is  removed,  and  when 
hard  enough  for  the  purpose,  sections  are  made  from  the  brown 
surface,  placed  in  water,  and  mounted  in  balsam.  The 
pigment-cells  usually  obscure  the  silver  markings  to  a  certain 
extent.  This  inconvenience  can  be  obviated  by  using  the  eye 
of  an  albino  rabbit.  Here,  moreover,  the  sclerotic  is  not  too 
thick  to  admit  of  the  piece  being  mounted  entire,  two  or  three 
radial  slits  being  made  in  it  if  necessary,  with  the  object  of 
causing  it  to  lie  flat  on  the  slide. 

Besides  this  preparation  of  its  epithelioid  layer,  the  lamina 
fusca  may  itself  be  displayed  in  an  eye,  or  portion  of  an  eye, 
that  has  been  prepared  with  bichromate.  To  obtain  it,  a 
small  piece  of  the  sclerotic  is  pinned  to  a  cork  or  wax-cake 
under  weak  spirit  (equal  parts  of  water  and  spirit) ;  and  the 
lamina  fusca  is  dissected  off  from  its  inner  surface,  and  floated 
on  to  a  slide,  the  spirit  being  then  allowed  to  evaporate,  so  as 
to  leave  the  delicate  membrane  moistened  only  with  water. 
The  preparation  may  now  be  covered,  and  glycerine  added  at 
the  edge  of  the  cover-glass. 

Sections  of  cornea. — The  several  layers  of  which  the 
cornea  is  composed,  and  their  relative  thickness,  should  first 


THE  EYE  201 

be  studied  in  sections  made  vertically  to  its  surfaces.  For 
this  purpose  the  anterior  part  of  an  eye  (pig's  or  ox's  if  a 
human  eye  is  not  procurable)  is  placed  in  3  per  cent,  solution 
of  bichromate  of  potash  or  jMiiller's  fluid  for  two  or  three 
weeks,  or  in  10  p.c.  formol  for  a  few  days.  It  is  as  well 
to  remove  the  lens  so  that  the  solution  passes  freely  to 
the  posterior  surface  of  the  cornea.  After  the  time  speci- 
fied the  tissue  is  put  into  weak  spirit  for  twenty-four  hours, 
and  then  transferred  to  strong  spirit.  In  tAvo  or  three 
days  more  it  will  be  ready  for  making  sections.  For  this 
purpose  a  piece  of  the  cornea  is  cut  out  and  embedded  in 
paraffin.  Very  thin  sections  vertical  to  the  surface  are  to  be 
made,  and  stained  and  mounted  in  the  usual  way  in  balsam. 
Sections  can  also  be  easily  made  by  the  freezing  method,  and 
after  being  stained  with  htymalum  or  carmalum  mounted  in 
balsam.  In  this  process  a  source  of  difficulty  is  sometimes 
met  with,  in  the  curling  up  of  the  posterior  part  of  the  section 
when  transferred  from  spirit  to  oil  of  cloves.  This  can  some- 
times be  got  rid  of,  without  spoiling  the  section,  by  careful 
manipulation  with  needles ;  but,  if  it  be  found  impossible  to 
obviate  it  in  any  other  way,  the  plan  may  be  adoj^ted  of 
placing  each  section,  after  it  has,  as  usual,  been  stained  and 
rinsed  in  water,  in  absolute  alcohol  for  a  few  minutes,  trans- 
f en'ing  it  to  a  slide,  and  immediately  covering  it  with  the  thin 
glass.  Oil  of  cloves  is  then  allowed  to  run  under  and  clarify 
the  specimen,  wliich  is  prevented  from  curling  up,  in  conse- 
quence of  the  pressure  of  the  cover-glass.  These  precautions 
are  not  necessary  in  sections  which  have  been  made  from 
paraffin  and  fixed  to  the  slide. 

Epithelium  of  the  cornea. — The  stratified  epithelium 
covering  the  front  of  the  cornea  is  well  seen  in  the  vertical 
sections,  but  the  characters  of  the  individual  cells  which  com- 
pose it  must  be  studied  in  a  teased  preparation.  For  this 
purpose  a  piece  of  the  cornea  is  placed  in  a  comparatively 
large  quantity  of  j^  per  cent,  bichromate  of  potash  solution  or 
one-third  alcohol,  and  allowed  to  remain  in  this  for  a  week. 


262  PKACTICAL   HISTOLOGY 

Then,  with  the  point  of  a  scalpel  or  spear-headed  needle,  a 
small  portion,  including  however  the  whole  thickness  of  the 
epithelium,  is  scraped  off  the  front,  placed  in  a  drop  of  distilled 
water  on  a  slide,  and  broken  up  with  needles  as  finely  as 
possible.  A  piece  of  hair  is  added,  and  lastly  the  cover-glass, 
and  the  specimen  is  then  ready  for  examination.  The  cells  of 
the  various  layers  will  be  recognised  by  the  characteristic 
forms  they  present ;  those  of  the  deepest  layer  being  in  shape 
like  a  rifle-bullet,  those  next  above  cupped  to  receive  the 
rounded  or  conical  ends  of  the  deeper  cells,  and  the  super- 
ficial layers  being  more  flattened  as  they  are  nearer  the 
surface.  The  fine  ridges  and  spines  on  many  of  the  cells  can 
be  distinctly  made  out  with  a  high  power,  and  give  a  jagged 
contour  to  the  cell. 

To  preserve  the  preparation  permanently  in  glycerine  it 
should  first  be  stained.  This  is  readily  done  by  applying  a 
drop  of  a  very  weak  solution  of  carmalum  to  the  edge,  and 
allowing  it  to  diffuse  under  the  cover-glass  ;  after  a  short 
time  glycerine  is  added  at  the  same  edge,  and  gradually 
replaces  the  stain,  the  water  from  which  evaporates  meanwhile 
at  the  other  borders  of  the  cover-glass. 

The  substantia  propria  of  the  cornea. — The  fibrous  struc- 
ture of  the  substantia  propria  of  the  cornea  can  readily  be 
seen  by  teasing  out  either  a  fresh  cornea,  or  one  which  has 
been  macerated  for  a  while  in  weak  spirit  or  in  picric  acid. 
The  lamellge  which  ■  the  fibrous  bundles  form  are  apparent 
when  an  attempt  is  made  to  tear  the  corneal  tissue,  and  they 
are  well  seen,  cut  in  different  directions,  in  the  vertical 
sections  previously  made. 

The  corneal  corpuscles  are  visible  in  the  sections  as  mere 
lines,  each  with  an  enlargement  in  the  middle,  stretching 
across  the  containing  cell-space,  which  is  fusiform  in  section 
and  is  seldom  filled  by  the  included  corpuscle.  These  appear- 
ances are  well  observed  in  the  human  cornea,  and  may  also  be 
made  out  in  that  of  the  pig  and  those  of  other  animals.  But 
although  they  look  like  mere  lines  in  vertical  section,  they 


THE   EYE  263 

are,  like  most  other  connective  tissui-  cells,  in  reality  flattened 
out  conformably  to  the  surfaces  of  the  lamellie,  and  present 
when  viewed  flat  great  in-egularities  of  form,  and  numerous 
branching-  processes  with  which  they  come  in  many  cases  into 
connection  with  one  another.  They  are  best  brought  to  view  l)y 
the  gold  method,  and,  since  this  also  serves  to  show  the  nerves, 
the  two  structures  may  be  studied  in  the  same  preparation. 

Corpuscles  and  nerves  of  the  frog's  cornea. — The  brain 
and  spinal  cord  of  a  frog  having  been  destroyed,  the  animal 
is  laid  on  the  table  or  held  by  an  assistant,  and  the  membrana 
nictitans  of  the  eye  is  seized  with  forceps,  and  entirely 
removed  by  two  or  three  snips  with  fine  sharp-pointed  scissors. 
The  animal  is  then  taken  up  and  held  in  the  operator's  left 
hand,  the  thumb  pressing  upwards  under  the  lower  jaw,  so 
that  the  eyes  are  made  as  prominent  as  possible,  and  the  point 
of  one  of  the  scissor  blades  is  inserted  into  the  globe  of  the 
eye,  just  behind  the  insertion  of  the  glistening,  yellowish  iris. 
By  a  series  of  snips  made  round  the  eyeball  at  this  plane,  the 
anterior  part,  with  the  cornea,  iris  and  lens,  is  severed  from 
the  posterior,  and  removed  to  a  watch-glass  containing  salt 
solution.  Then,  whilst  the  edge  of  the  cut  sclerotic  is  held  by 
the  one  pair  of  forceps,  with  another  pair  the  iris  is  seized 
close  to  the  same  spot,  and  is  easily  torn  away  from  the 
sclerotic,  bringing  the  lens  with  it.  So  that  only  the  cornea, 
together  with  a  narrow  rim  of  sclerotic,  is  now  left,  and  since 
it  is  floating  in  fluid  it  retains  its  convexo-concave  form,  and 
all  crumpling  of  the  tissue  is  avoided.  The  salt  solution  is 
now  poured  off,  leaving,  however,  just  enough  to  float  the 
cornea  in,  and  the  watch-glass  is  filled  up  with  ^  per  cent, 
chloride  of  gold  solution.  The  cornea  is  allowed  to  remain  in 
this  a  full  hour  ;  it  is  then  removed  to  a  beaker  of  water 
acidulated  with  acetic  acid,  and  is  placed  in  a  wai-m  place  in 
the  sunlight  (see  p.  120).  After  two  days  the  fluid  in  the 
beaker  is  rene%\ed,  a  teaspoonful  of  methylated  spirit  being 
added  to  prevent  the  growth  of  fungi,  and  in  two  days  more 
the  cornea  may  be  taken  out  and  prepared  for  the  microscope. 


264  PEACTICAL  HISTOLOGY 

It  is  first  placed  in  a  flat  dish  of  distilled  water,  and  the  epi- 
theliuDQ,  which  is  very  dark  and  opaque,  is  gently  scraped  off 
the  anterior  surface.  This  done,  the  sclerotic  rim  is  cut  off 
with  scissors.  It  is  as  well  to  change  the  water  at  this  stage, 
so  as  to  get  rid  of  the  debris  of  epithelium.  The  next  process 
consists  in  the  separation  of  the  corneal  substance  into  two, 
three,  or  more  thin  lamellge.  With  a  little  practice  it  is  not 
at  all  difficult,  thin  as  the  object  already  seems,  by  holding  it 
down  at  one  edge  with  a  pair  of  forceps  and  working  from  the 
same  edge  with  another  pair,  to  separate  a  very  thin  lamina 
from  the  concave  posterior  surface,  consisting  of  the  membrane 
of  Descemet  and  a  delicate  layer  of  the  proper  substance  of  the 
cornea  with  its  corpuscles.  This  posterior  lamella  is  not  only 
the  easiest  to  obtain,  but  is  also,  in  the  frog's  cornea,  the 
most  important,  for  it  contains  the  closest  and  finest  plexus 
of  nerves.  To  mount  it,  all  that  is  necessary  is  to  float  it  on 
to  a  glass  slide,  to  cover  the  preparation,  and  add  glycerine 
at  the  edge  of  the  cover-glass.  But,  since  the  membranous 
layer  thus  obtained  has  naturally  the  convex  shape  of  the 
cornea,  and  it  is  of  course  desirable  that  it  should  lie  flat 
upon  the  slide  without  creases,  it  is  well  before  mounting 
to  make  three  or  four  radial  snips  in  it  in  the  way 
shown  in  the  adjoining  cut  ;  these  will  enable  it  to 
flatten  out  without  folding  when  placed  on  the  slide 
and  covered.  Moreover  it  is  important  to  examine 
the  object '  with  a  low  power  previously  to  covering 
it,  so  that  any  folds  of  the  membrane,  or  any  foreign  matter 
or  remains  of  epithelium  adhering  to  it,  may  be  detected  and 
removed. 

The  remaining  anterior  part  of  the  cornea  may  be  further 
separated  in  the  same  way  into  lamellte,  which  are  to  be 
mounted  with  the  same  precautions  as  the  posterior  lamella. 
It  is  not  always  easy  to  get  them  in  quite  so  complete  a  layer, 
but  for  most  purposes  a  small  shred  will,  if  equally  thin,  show 
nearly  as  much  as  an  entire  lamella. 

In  all  these  specimens  the  corneal  corpuscles  are  stained  of 


THE   EYE  265 

a  violet  colour,  varying  in  tint  according  to  the  success  of  the 
preparation,  their  nuclei  being  left  nearly  unstained.  The 
nei'ves  are  coloured  almost  black,  the  fibrils  looking  like  fine 
wires  running  singly  and  in  bundles,  and  provided  along  their 
course  with  numerous  minute  varicosities. 

The  corpuscles  and  nerves  of  the  rabbit's  cornea. — The 
cornea  of  the  rabbit,  or  of  any  other  mammal  recently  killed, 
is  prepared  with  chloride  of  gold  in  the  same  way  as  that  of 
the  frog.  The  eyelids  are  first  removed,  care  being  taken  in 
doing  this  not  to  get  the  hair  on  to  the  surface  of  the  cornea. 
The  eye  is  then  to  be  made  prominent,  either  by  an  assistant 
who  holds  it  firmly  with  forceps  thrust  back  in  the  orbit,  so 
as  to  seize  one  or  other  of  the  eye  muscles  near  their  attach- 
ment, or,  without  an  assistant,  by  clamp-forceps,  which  are 
inserted  in  like  manner,  and  by  their  weight  force  the  eyeball 
forward  without  unduly  compressing  it.  Then  the  cornea, 
iris,  and  lens  are  removed  together,  after  cutting  round  the 
sclerotic  with  scissors,  and  are  placed  in  salt  solution,  and 
finally  the  iris  and  lens  are  removed  in  the  same  way  as  in  the 
preceding  preparation,  and  the  cornea  is  immersed  in  gold 
solution.  Since  it  is  much  thicker  than  the  frog's  cornea,  it 
should  remain  in  the  chloride  of  gold — which  does  not  pene- 
trate very  rapidly — a  full  hour  and  a  half,  after  which  it  is 
placed  in  acidulated  water  in  the  light,  and  otherwise  treated 
in  the  same  way  as  the  frog  cornea.  But  as  it  is  not  so  easy 
to  separate  the  mammalian  cornea  into  lamelhe,  it  is  better 
after  three  or  four  days  to  place  the  stained  cornea  in  gum  for  a 
few  hours,  when  thin  sections  parallel  to  the  surface  may  be 
cut  with  the  freezing  microtome,  and  mounted  in  glycerine. 

Isolation  of  corneal  corpuscles. — After  the  corneal  cor- 
puscles and  nerves  have  been  stained  with  gold  in  this  way, 
they  can  be  isolated  by  dissolving  away  the  intermediate  sub- 
stance by  caustic  alkali,  the  action  being  arrested  before  the 
corpuscles  and  nerves,  which  are  more  resisting  than  the 
connective  tissue  bundles,  are  also  destroyed.  With  this 
object  a  part  or  the  whole  of  a  gold-stained  cornea — whether 


266  PEACTICAL  HISTOLOaY 

of  frog  or  mammal — is  placed,  divested  of  epithelium,  in  a 
■watch-glass  containing  a  strong  (20  per  cent.)  solution  of 
caustic  potash  or  soda,  and  this  is  then  put  into  a  warm 
chamber  at  40°  C.  At  the  expiration  of  three-quarters  of  an 
hour  the  tissue,  which  is  now  quite  soft  and  pulpy,  is  removed 
with  a  section-lifter,  and  placed  in  a  vessel  containing  a  large 
quantity  of  water  faintly  acidulated  with  acetic  acid.  Small 
portions  may  then  be  taken  up  and  mounted,  with  or  without 
further  breaking  up,  in  glycerine.  The  corpuscles  are  beauti- 
fully displayed,  forming  a  continuous  network  by  the  ju.nction 
of  their  branches  ;  and  nervous  fibrils  may  be  seen  interca- 
lated amongst  the  corpuscles,  but  never  actually  joined  to,  or 
continuous  with,  the  cells. 

Nerves  of  the  rabbit's  cornea. — For  exhibiting  the  nerves 
of  the  cornea  without  at  the  same  time  staining  the  corpuscles 
and  the  epithelium,  either  Golgi's  silver  chromate  method 
(p.  152),  or  Ehrlich's  methyl-blue  method  (p.  160),  or  Lowit's 
gold  chloride  method  (p.  159),  or  the  following  modification 
of  the  gold  method  recommended  by  Klein,  may  be  used. 
The  cornea  of  a  rabbit  or  guinea-jDig  is  put  irito  ^  per  cent, 
solution  of  chloride  of  gold  for  an  hour  and  a  half.  It  is 
removed  from  the  gold  into  distilled  water  and  placed  in 
the  light  (without  warmth)  for  from  twenty-four  to  thirty 
hours,  or  until  the  larger  nerve  trunks  begin  to  be  visible 
near  the  circumference,  converging  towards  the  centre  as 
irregular,  branching  lines.  When  the  staining  has  arrived 
at  this  stage,  and  before  the  corneal  substance  generally  begins 
to  acquire  a  violet  appearance,  the  cornea  is  removed  from  the 
water  and  placed  in  a  mixture  of  glycerine  and  water  (one 
part  glycerine  to  two  parts  distilled  water).  After  it  has  been 
in  this  for  twenty-four  hours,  or  longer,  the  corneal  substance 
should  be  very  little  darker  than  before,  but  the  nerves  much 
more  distinct,  and  on  holding  the  cornea  between  the  finger 
and  thumb,  and  making  sections  from  the  anterior  sui'face, 
including  the  epithelium  and  a  little  of  the  substantia  propria, 
these,  when  covered  in  the  glycerine  mixture,  will  show  not 


THE   EYK  207 

only  the  due  and  close  plexus  of  nerves  which  lies  immediately 
underneath  the  epithelium,  but  also  the  fai'  more  minute 
network  of  varicose  ultimate  fibrils  which  extends  between 
the  einthelium  cells  almost  to  the  anterior  surface  of  the 
epithelium.  If  not  at  first  sufficiently  evident,  these  intra- 
epithelial nerves  may  generally  be  brought  more  clearly  into 
view  by  placing  a  section  for  a  few  minutes  in  the  strong 
caustic  potash  solution.  From  this  it  is  transferred  by  a  sec- 
tion-lifter to  water,  floated  on  to  a  slide,  removed  from  the 
water,  and  covered,  glycerine  being  afterwards  added  at  the 
edge  of  the  cover-glass.  To  prevent  the  latter  from  pressing 
on  the  softened  tissue,  two  narrow  slips  of  thin  glass,  which 
may  be  cut  with  a  writing  diamond,  are  to  be  placed  one  on 
either  side,  Ijefore  placing  the  cover-glass  over. 

The  cell-spaces  of  the  cornea.-  These  are  shown  in  two 
ways  :  by  the  silver  method,  and  by  the  method  of  puncture 
injection. 

The  demonstration  of  the  cell-spaces  by  the  silver  method 
may  be  attempted  in  the  cornea  of  the  frog.  The  animal  is 
decapitated  and  the  brain  destroyed.  The  eyelid  is  then 
removed,  and  the  eye  having  been  made  prominent  by  the 
pressure  of  the  thumb  in  the  manner  previously  recommended, 
the  epithelium  is  scraped  oft'  the  front  of  the  cornea  with  a 
sharp  scalpel.  The  cornea  is  then  rubbed  with  a  stick  of 
fused  nitrate  of  silver  (lunar  caustic).  After  five  minutes  the 
surface  is  thoroughly  washed  with  a  stream  of  distilled  water 
from  a  wash-bottle.  The  head  is  now  placed  in  spirit  in  the 
light  ;  in  a  short  time  (from  a  few  minutes  to  an  hour),  when 
the  cornea  is  browned,  the  vessel  containing  it  is  removed 
from  the  window  and  left  in  a  dark  place  for  twenty- four 
hours.  The  cornea  is  then  sliced  off",  placed  in  water,  where 
any  remaining  patches  of  epithelium  are  now  removed  by 
scraping,  slit  in  a  triradiate  manner,  so  that  it  may  lie  flat  on 
a  slide,  and  finally  mounted  in  balsam. 

The  cornea  of  mammals  may  be  prepared  with  silver  in  a 
similar  mannei*,    but,   being  genei'ally   thicker    than  in    the 


268  PEACTICAL  HISTOLOGY 

frog,  it  is  necessary  to  allow  the  caustic  a  longer  time  to 
penetrate,  and,  in  the  final  preparation,  to  prepare  sections 
parallel  to  the  surface,  instead  of  mounting  the  cornea 
whole. 

To  inject  the  cell-spaces  of  the  cornea  by  the  puncture 
method,  the  eye  of  the  pig,  or  sheep,  or  any  other  animal 
may  be  used,  if  a  human  eye  is  not  procurable  for  the  purpose. 
A  solution  of  alkanet  in  turpentine  is  the  fluid  which  should 
be  chosen,  and  the  mercurial  pressure  apparatus  (fig.  56)  is 
used.  The  tvxbe  and  fine  steel  cannula  having  been  filled 
with  the  alkanet  solution  to  the  exclusion  of  air-bubbles,  the 
cannula  is  inserted  obliquely  into  the  substance  of  the  cornea, 
without  allowing  the  point  to  pass  through  into  the  anterior 
chamber.  The  px"essure  is  then  gradually  raised  to  about  two 
inches  of  mercury,  when  the  red  fluid  should  begin  gradually 
to  fill  the  cell-spaces,  and  to  spread  through  them  over  a  con- 
siderable part  of  the  cornea.  Indeed,  if  the  injection  is  long 
enough  continued,  the  fluid  may  extend  itself  even  beyond 
the  corneal  margin,  and  may  penetrate  into  the  cell-spaces  in 
the  anterior  part  of  the  sclerotic  coat.  The  operation  may 
with  care  be  successfully  performed  without  the  mercurial 
apparatus,  using  merely  a  hypodermic  syringe.  But  it  is  diffi- 
cult to  avoid  the  production  of  extravasation  near  the  point 
of  the  cannula.  This  does  not,  however,  always  militate 
against  the  success  of  the  experiment,  for  beyond  the  limit 
of  the  extravasation  the  duid  may  slowly  penetrate  into  the 
cell-spaces  of  the  tissue,  and  this  may  go  on  even  after  the 
syringe  has  been  removed,  especially  if  the  cornea  is  cut  out, 
laid  flat  on  a  slide,  and  allowed  to  dry.  As  watery  fluid  be- 
comes withdrawn  from  the  cell-spaces  in  the  process  of  diying 
the  alkanet  solution  tends  to  pass  in  to  occupy  its  place. 

Another  very  successful  plan  of  inducing  the  injecting 
fluid  to  pass  from  such  an  extravasation  into  the  neighbouring 
cell-spaces  consists  in  gently  stroking  from  the  extravasation 
towards  the  margin  of  the  cornea  with  a  smooth  instrument, 
such  as  a  glass  rod  or  the  ivory  handle  of  a  scalpel.     But  if 


THE   EYE  2G9 

too  great  pressure  is  exerted  upon  the  lluiil  it  will  be  found 
that,  in  place  of  taking  the  closely  reticulating  course  which 
it  would  pursue  if  it  merely  occupied  the  cell-spaces,  the 
injection  tends  to  shoot  through  the  tissue  in  straight  lines 
which  in  successive  planes  of  the  corneal  tissue  run  at  right 
angles  to  one  another.  These  lines  represent  the  'corneal 
tubes  '  of  Bowman.  Their  appearance  is  due  to  the  fact  that 
the  pressure  exerted  has  been  sufficient  to  force  the  injecting 
fluid  into  the  interstices  between  the  connective  tissue  Ijundles, 
pushing  these  asunder,  and  burrowing  its  way  through  the 
soft  ground-substance  which  unites  the  bundles  and  lamella?. 
And,  since  the  cell-spaces  occur  in  this  ground-substance,  the 
existence  of  a  slight  enlargement  or  fusiform  swelling  here 
and  there  on  the  tubes  is  accounted  for. 

These  corneal  tubes  then  are  to  be  looked  upon  as  purely 
artificial  products,  not  corresponding  with  any  pre-existing 
channels  in  the  tissue  (except  perhaps  when  the  fluid  passes 
along  the  sheath  of  a  nerve).  They  are  always  obtained  when 
any  fluid  which  is  not  able  to  penetrate  into  the  cell-spaces  is 
forced  into  the  substance  of  the  cornea.  They  are  seen  when 
mercury  is  mjeeted  by  the  puncture  method,  and  this  is  how 
they  were  first  obtained  by  Bowman  ;  and  if  air  be  forced 
with  a  syringe  into  the  tissue  a  similar  effect  is  produced.  In 
all  cases  the  tubes  cease  abruptly  at  the  corneo-sclerotic  junc- 
tion, where  the  connective  tissue  becomes  denser,  and  has  a 
less  regular  arrangement. 

Parts  at  the  junction  of  the  cornea  with  the  sclerotic. — 
The  corneo-sclerotic  junction,  the  ciliary  muscle,  and  the  iris 
are  all  well  seen  in  their  relations  to  one  another  in  a  meridional 
section  of  the  part  of  the  eye  where  they  are  situated.  The  sec- 
tion may  be  made  from  the  anterior  segment  of  an  eye  hardened 
in  potassium  bichromate  or  in  5  to  10  per  cent,  formol.  It  is 
not  necessary  to  imbed  this  entire,  but  sufficient  to  cut  out  with 
sharp  scissors  under  spirit  a  piece  which  includes  all  the  parts 
above  enumerated.  The  piece  is  cut  by  either  the  collodion 
or  the   paraffin   method.     Few   specimens   better   repay   the 


270  PEACTICAL  HISTOLOGY 

trouble  of  prepai-ation  than  these.  The  cornea,  sclerotic,  iris, 
choroid,  ligamentum  pectinatum,  canal  of  Schlemm,  ciliary 
muscle,  both  radial  and  circular,  and  even  the  ora  serrata  and 
pars  ciliaris  of  the  retina,  are  all  exhibited  with  the  greatest 
clearness  in  a  successful  section,  and  their  structure  and  rela- 
tions may  be  advantageously  studied. 

To  see  the  glandular  depressions  of  the  pigmented 
epithelium  covering  the  choroid  processes  it  is  necessary  to 
remove  the  pigment  from  a  section  by  bleaching.  This  is 
eflFected  by  the  action  of  euchlorine,  chlorine  water,  or  solution 
of  hypochlorite  of  soda. 

Ciliary  muscle  and  lamina  suprachoroidea. — To  prepare 
these,  the  anterior  part  of  an  eye  hardened  in  Miiller's  fluid 
is  pinned  down  under  spirit,  and  the  cornea  and  sclerotic  cut 
away  at  one  part,  when  the  radiating  fibres  of  the  ciliary 
muscle  will  be  seen  passing  meridionally  from  their  origin 
opposite  the  attachment  of  the  iris,  and  forming  a  layer  which 
becomes  gradually  thinner  as  it  extends  backwards  and  finally 
ceases  in  the  superficial  part  of  the  choroid.  A  small  piece  of 
the  muscle  is  seized  with  sharp  forceps  near  its  anterior  border 
and,  by  carrying  the  instrument  slowly  backwards,  is  gradually 
torn  away  from  the  rest.  It  will  be  found  that  the  shred 
which  comes  away  generally  spreads  out  posteriorly  into  a 
very  thin  membranous  lamina,  this  being  in  fact  a  piece  of 
the  lamina  suprachoroidea  into  which  the  superficial  fibres  of 
the  ciliary  muscle  are  inserted.  A  considerable  length  may 
be  torn  off  in  this  way,  and  the  piece  so  obtained  is  to  be 
floated  directly  on  to  a  slide,  which  is  dipped  for  the  pui^pose 
into  the  spirit.  It  must  be  moved  with  great  care,  so  as  to 
avoid  folds  or  creases.  The  slide  is  quickly  wiped  free  of  spirit 
with  the  exception  of  that  which  immediately  moistens  the  speci- 
men, and  a  drop  of  hsemalum  or  picrocarmine  solution  is  placed 
upon  the  tissue,  and  allowed  to  remain  on  it  for  ten  minutes, 
or  sufficiently  long  to  impart  its  colour  to  the  tissue.  The 
staining  solution  is  then  poured  off,  and  the  remains  of  it 
are  removed  by  allowing  a  drop  or  two  of  water  to  flow  gently 


THE   EYE  271 

over,  without  disturbing  the  position  of  the  membrane.  Finally 
a  cover-glass  is  laid  on,  and  a  drop  of  glycerine  allowed  ti)  run 
in  at  the  edge  of  the  cover-glass. 

The  preparation  so  obtained  is,  if  successful,  a  very 
striking  one.  Besides  the  branched  pigment  cells  of  the 
choroidal  tissue,  and  a  certain  number  of  cells,  similar  to 
white  blood-coi'puscles,  on  the  surface  of  the  membrane,  a 
number  of  large,  round  or  oval  nuclei  are  seen  in  the  lamina, 
which  are  apparently  devoid  of  cell-body.  These  are  the 
nuclei  of  epithelioid  cells  which  bound  the  lamina  supra- 
chovoidoa  externally,  and  serve  as  part  of  the  lining  of  the 
lymphatic  space  which  lies  between  this  and  the  lamina  f  usca 
of  the  sclerotic.  Their  outlines  cannot  be  brought  to  view 
without  the  aid  of  nitrate  of  silver,  and  the  cell-bodies  are 
too  delicate  and  transparent  to  be  shown  by  the  present 
mode  of  preparation.  A  large  number  of  elastic  fibres  are 
also  seen  on  the  membrane,  especially  at  the  terminations  of 
the  fibres  of  the  ciliary  muscle,  where  they  appear  to  come 
into  relation  with  the  ends  of  these,  an  elastic  fibre  passing 
for  a  certain  distance  along  each  side  of  the  muscular  filire, 
and  seeming  to  serve  in  this  way  for  its  attachment.  The 
involuntary  fibres  are  particularly  well  shown,  their  nuclei 
being  conspicuously  stained  by  hsematein ;  many  of  the 
bundles  terminate  in  peculiar  tufts,  from  which  the  fibres 
radiate  in  all  directions.  It  may  happen  that  the  preparation 
includes  one  of  the  long  ciliary  nerves ;  this,  as  it  coursed  for- 
wai'd  to  enter  the  ciliary  muscle,  having  been  torn  away 
with  the  shi'ed  of  membrane.  If  so,  it  may  be  followed  with 
the  microscope  and  its  branches  traced  amongst  the  bundles 
of  muscular  fibres,  forming  a  plexus  with  those  of  the  other 
nerves.  In  tracing  the  branches  characteristic  bipolar 
ganglion- cells  will  here  and  there  be  found  interpolated  in  the 
course  of  the  nerve-fibres. 

Vascular  layers  of  choroid  and  membrane  of  Briich. — 
As  seen  in  the  preparation  just  described,  it  is  easy  to  detach 
the    lamina   suprachoroidea    from    the    rest   of   the    choroid. 


272  PEACTICAL  HISTOLOGY 

The  other  three  parts  are  more  difficult  to  separate,  and 
their  complete  isolation  may  require  considerable  time  and 
patience.  But  for  demonstrating  their  structure  it  is  not 
absolutely  necessary  for  them  to  be  completely  separated  as 
distinct  membranes ;  it  is  sufficient  if,  in  a  piece  which  in- 
cludes all,  one  or  other  is  left  projecting  at  the  edge,  so  as  in 
this  way  to  be  seen  distinct  from  the  other  layers.  But  before 
commencing  the  attempt  at  separation,  the  hexagonal  pigment 
cells,  which  belong  to  the  retina  but  frequently  adhere  to  the 
inner  surface  of  the  choroid,  must  be  entirely  removed  by 
gently  brushing  that  surface  with  a  hair-pencil.  The  separation 
and  brushing  are  performed  under  fluid  (spirit),  and  will  be 
much  facilitated  by  the  use  of  a  dissecting  lens. 

The  musculature  of  the  iris. — The  circular  and  radiating 
plain  muscular  fibres  of  the  iris  may  be  demonstrated  in  the 
albino  rabbit.  The  eye  is  cut  in  half  and  the  anterior  part 
placed  in  spirit  for  a  day  or  more.  Then  the  lens  is  removed, 
and  a  segment  of  the  iris — including  its  whole  width,  from  the 
pupillary  aperture  to  the  ciliary  processes  of  the  choroid — is 
cut  out  and  placed  in  dilute  haemalum.  When  sufficiently 
but  not  too  deeply  stained,  it  is  put  into  water  for  a  minute 
or  two  to  remove  the  excess  of  staining  fluid,  then  passed 
through  picric  alcohol  and  oil  of  cloves,  and  finally  mounted  in 
balsam,  with  the  posterior  surface  uppermost.  The  thick  ring 
of  the  sphincter  is  easily  seen  in  these  preparations,  and  also  the 
interlacing  bundles'  of  plain  muscular  fibres  of  which  the 
dilatator  is  composed  :  they  may  be  obser^^ed  to  bend  round 
near  the  pupil,  and  take  the  direction  of  and  blend  with  those 
of  the  sphincter.  At  the  circumference  of  the  iris,  also,  a 
similar  bending  round  of  the  radiating  fibres  is  observed. 

Human  iris. — Although,  in  consequence  of  the  presence 
of  the  uveal  pigment,  more  troublesome  to  prepare,  it  is 
nevertheless  desirable  to  make  a  similar  preparation  of  the 
human  iris,  for  the  musculature  is  somewhat  diflerent,  the 
dilatator  forming  a  uniform  thin  expansion,  which  covers  the 
posterior  surface  immediately  under  the  pigmented  epithelium, 


THE   RETINA  273 

and  not  distinct  radially  arranged  interlacing  bundles  with 
intervening  meshes  as  in  the  rabbit.  The  specimen  may  be 
made  from  an  eye  that  has  been  in  Miiller's  Huid  or  bichro- 
mate of  potash  C-i  per  cent.),  preferably  not  in  spirit.  A 
piece  is  cut  out  as  before,  and  is  treated  in  a  similar  "way, 
except  that  before  being  stained  the  pigment  is  brushed  com- 
pletely otf  the  posterior  surface  with  a  stiff  camel-hair  brush. 
This  must  be  done  under  fluid,  and  of  course  very  carefully 
so  as  to  avoid  tearing  the  tissue  ;  during  the  operation  the 
iris  is  examined  now  and  then  with  a  low  power,  to  determine 
when  all  the  pigment  is  removed.  It  is  difficult  to  prevent 
some  of  the  pigment  granules  from  still  sticking  to  the  surface, 
but,  as  they  tend  for  the  most  part  to  adhere  along  the  lines 
of  junction  of  the  tibre-cells,  their  presence  does  not  spoil  the 
object  of  the  preparation,  for  the  dilatator  fibres  are  if  any- 
thing better  displayed. 

A  method  of  getting  rid  of  the  remains  of  the  pigment 
is  to  treat  the  piece  of  iris,  after  brushing,  with  chlorine  water 
or  solution  of  hypochlorite  of  soda  until  completely  bleached  : 
then  wash,  stain,  and  mount  as  above. 

THE    RETINA 

Sections  of  the  retina. — It  will  be  well  to  study  the 
general  arrangement  of  the  several  layers  of  the  retina  by 
means  of  sections,  before  its  constituent  elements  are  observed 
isolated.  The  best  method  of  hardening  is  by  a  mixture  of 
three  parts  Midler's  fluid  and  one  part  of  1  per  cent,  osmic 
acid,  the  process  being  completed  by  spirit.  Corrosive 
sublimate  may  also  be  used.  If  possible  to  obtain  it  perfectly 
fresh  and  healthy,  the  human  retina  should  always  be  taken  ; 
if  not  so  obtainable,  that  of  the  pig  is  preferable  to  the  retina 
of  most  other  common  mammals..  For  the  study  of  the 
central  fovea,  the  human  eye  or  that  of  a  monkey  is 
necessary. 

To  harden  the  retina  rather  small  pieces  are  cut  out  from 

T 


274  PEACTICAL  HISTOLOGY 

different  parts,  or,  what  is  better,  the  posterior  part  of  the 
bulb  is  kept  whole,  so  that  the  membrane  remains  supported 
by  the  outer  coat.  This  posterior  part  should  be  turned 
inside  out  and  the  vitreous  removed,  and  it  is  then  dropped 
into  a  relatively  large  quantity  of  the  hardening  fluid,  and 
allowed  to  remain  in  it  for  a  week,  the  fluid  being  stirred  at 
intervals  and  changed  on  the  second  day.  After  a  week  it  is 
transferred  first  for  two  or  three  hours  to  water,  then  for 
twenty-four  hours  to  50  per  cent,  alcohol,  and  then  to  strong 
spirit.  In  another  day  or  two  it  is  ready  for  the  preparation 
of  sections.  Small  pieces  from  different  regions  may  be  taken, 
but  as  they  are  all  treated  in  the  same  way  they  may  be 
described  as  one. 

The  staining  of  the  tissue  is  first  effected  by  placing  the 
piece  for  twenty-four  hours  in  alcoholic  logwood  (Kleinen- 
berg's)  or  alcoholic  fuschin.  From  this  it  is  transferred  to 
spirit,  then  embedded  in  paraffin.  It  is  better  so  to  place  it 
in  the  embedding  tray  that  the  sections  shall  be  both  vertical 
and  meridional,  since,  made  in  this  way,  they  will  take  the 
general  course  of  the  fibres  of  the  optic  nerve.  The  sections 
cannot  be  made  too  thin,  but  they  should  be  complete,  that  is 
to  say,  they  should  include  all  the  layers  of  the  membrane. 
They  are  cut  in  series,  fixed  with  water  (p.  37)  and  mounted 
in  balsam.  But  if  hardened  with  corrosive  sublimate  or  cor- 
rosive and  picric  they  are  best  stained  on  the  slide,  and  for  this 
any  of  the  ordinary  methods,  and  especially  methyl  blue  or 
toluidin  blue  and  eosin  (p.  23),  may  be  employed. 

Preparation  by  the  Golgi  method. — As  Cajal  has  shown, 
no  method  shows  the  relations  of  the  retinal  elements  to  one 
another  better  than  the  method  of  Golgi.  The  best  way  to 
employ  it  is  as  follows  : — Place  small  pieces  of  fresh  retina  from 
the  ox,  dog,  or  pig — preferably  pieces  which  are  rolled  up  upon 
themselves — in  a  relatively  large  quantity  of  the  osmium- 
bichromate  mixture  (one  part  1  per  cent,  osmic  to  three  parts 
Miiller)  :  keep  them  in  the  dark  for  from  one  to  five  days  : 
each  day  take  a  piece,  and  after  rinsing  it  in  Q'75  p.c.  nitrate 


THE   RETINA  275 

of  silver  solution  transfer  it  to  a  quantity  of  the  same  solution 
containing  a  trace  of  formic  acid  (1  drop  to  100  c.c). 
After  forty-eight  hours  in  the  silver  solution  wash  with  dis- 
tilled Avater,  transfer  to  95  per  cent,  alcohol  for  an  hour  or 
two  ;  place  in  collodion  for  a  few  minutes,  fix  on  a  metal 
holder  with  the  collodion,  and  immerse  in  95  per  cent,  alcohol  ; 
tlicn  cut  the  sections  with  a  sliding  microtome,  pass  them 
rapidly  through  absolute  alcohol  and  bergaraot  oil  or  xylol, 
and  mount,  without  a  cover-glass,  in  xylol  balsam.  A  large 
number  of  sections  should  be  mounted  in  this  way  and 
searched.  Some  will  show  only  Miillerian  fibres  ;  others  rod 
and  cone  elements  ;  others  the  inner  granules,  and  others  again 
the  nerve  elements  of  the  molecular  layer  :  all,  of  course, 
stained  black  by  the  reduced  metal. 

Ehrlich's  methylene-blue  method  (p.  160)  may  also  be  em- 
ployed for  retina,  with  great  advantage,  and  after  fixation  by 
Bethe's  fluid  followed  by  alcoholic  solution  of  platinic  chloride 
(1  in  300),  sections  may  be  made  by  the  paraffin  method. 

Isolation  of  the  retinal  elements. — Various  processes  are 
employed  for  macerating  portions  of  the  retina  in  order  to 
obtain  its  elements,  either  in  a  completely  isolated  condition 
or  still  partially  connected  with  one  another.  It  will  be  best, 
in  the  first  place,  to  attempt  the  separation  with  a  piece  of 
retina  which  has  been  in  1  or  2  per  cent,  osmic  acid  for  six  or 
eight  hours.  It  must  of  course  be  put  in  perfectly  fresh,  and 
after  the  time  mentioned  it  is  placed  in  water  for  twenty-four 
hours.  It  is  then  allowed  to  macerate  for  a  few  days  in  a 
mixture  of  equal  parts  of  glycerine,  alcohol,  and  water, 
after  which  a  minute  portion  is  to  be  carefully  broken  up  with 
fine  needles  in  a  drop  of  weak  glycerine,  and,  a  piece  of  tine 
hair  having  been  added,the  cover-glass  is  superposed  and  tapped 
to  effect  the  separation  and  isolation  of  the  elements.  The 
piece  of  retina  may  be  kept  for  years  in  the  alcohol  glycerine 
and  water  mixture,  and  the  longer  it  is  left  the  easier 
becomes  the  isolation. 

Other  portions  of  fresh  retina  may  be  placed  in  10  per 


276  PEACTICAL  HISTOLOGY 

cent,  chloral  hydrate  solution  for  two  or  three  days.  The 
portions  so  macerated  are  to  be  teased  out  in  a  drop  of  the 
same  solution,  the  usual  expedient  being  adopted  of  obviating 
the  pressure  of  the  cover-glass  by  a  hair. 

In  the  chloral  hydrate  preparation,  the  rod-  and  cone- 
elements  are  well  preserved,  as  are  also  most  of  the  other 
structures.  The  external  segments  of  the  rods,  which  even  in 
the  osmic  preparation  tend  for  the  most  part  to  become 
altered,  may  by  this  method  be  frequently  seen  almost 
unchanged,  and  the  transverse  striation,  which  indicates  their 
discoid  formation,  is  often  well  marked. 

The  study  of  the  retina  cannot  be  considered  complete 
until  the  elements  have  been  examined  in  the  fresh,  unaltered 
condition.  A  small  piece,  taken  from  an  eye  still  warm  from 
the  animal,  should  accordingly  be  broken  up  as  rapidly  and 
finely  as  possible  in  a  little  vitreous  humour.  A  modification 
of  this  very  simple  method  consists  in  allowing  a  small  piece 
to  macerate  for  two  or  three  days  in  weakly  iodised  serum 
(serum  or  amniotic  fluid  in  which  iodine  has  been  shaken  up) 
before  attempting  the  dissociation,  which  can  then  be  more 
readily  efiected. 

The  hexagonal  pigment  of  the  retina  may  be  seen  in  most 
of  the  teased  preparations  above  described.  In  eyes  that  have 
been  hardened  in  Miiller's  fluid  the  layer  often  separates  in 
flakes  of  varying  size,  and  nothing  is  simpler  than  to  remove  such 
a  piece  with  a  section-lifter,  and  mount  it  in  glycerine,  so  as 
to  exhibit  the  pavemented  appearance  which  the  cells  present. 

Silver  preparation. — On  the  innermost  surface  of  the  retina, 
also,  a  mosaic-like  appearance  can  be  demonstrated  by  the  aid 
of  nitrate  of  silver,  but  it  is  much  more  irregular,  and  does 
not  depend  upon  the  presence  of  epithelium  cells,  but  upon 
the  flattened-out  ends  of  the  Miillerian  fibres.  To  show  this 
appearance,  a  fresh  eye  is  cut  in  half  transversely,  and  the 
vitreous  is  shelled  out  from  the  posterior  half,  which  is  turned 
inside  out ;  this  is  then  rinsed  in  distilled  water  and  trans- 
ferred to  nitrate  of   silver  solution  (^   per   cent.).     After  a 


THE  RETINA  277 

minute  in  tliis  it  is  ;igain  rinsed  in  distilled  water,  and  exposed 
in  water  to  the  light.  When  the  retinal  surface  is  browned 
the  eye  is  removed,  a  piece  of  the  retina  is  cut  out  under  water, 
Heated  on  to  a  slide  with  the  inner,  brown  surface  uppermost, 
and  the  water  is  drained  off,  the  prepai-ation  allowed  to  di-y, 
and  then  mounted  in  balsam.  Or  it  may  be  mounted,  without 
drying,  in  glycerine,  care  being  taken  not  to  let  the  cover-glass 
press  upon  the  specimen. 

The  retina  in  the  lower  Vertebrata. — The  structure  of  the  outer 
segments  of  the  rods,  which  is  difficult  to  make  out  in  mammals, 
can  be  seen  easily,  even  with  an  ordinary  high  power,  in  the  retina 
of  amphibia.  "With  this  object  the  eye  is  removed  from  a  recently 
killed  frog,  cut  across,  and  a  small  portion  of  the  retina  is  quickly 
broken  up  in  vitreous  biunour.  A  piece  of  hair  having  been  added, 
the  preparation  is  covered  and  examined.  Almost  everywhere  the 
field  of  \ie\v  is  strewn  with  large,  clear,  rod-shaped  structures, 
some  straight,  but  many  of  them  bent  and  curved  in  different 
directions,  and  exhibiting  a  distinct  transverse  striation,  or  even  a 
tendency  to  split  up  into  a  number  of  superimposed  discs,  this 
tendency  increasing  as  the  preparation  is  longer  made.  Some  of 
them  have  what  looks  like  a  small  appendage  jointed  on  at  one 
end ;  this  is  the  comparatively  small  inner  segment  of  the  rod. 
The  cones  are  also  small  in  comparison,  and  on  that  account  may 
at  first  be  missed  ;  they  are  distinguished  by  the  possession  at  the 
apex  of  the  inner  segment  of  a  small,  bright,  fatty  globule,  often  of 
a  yellow  colour.  Most  likely  a  portion  of  the  hexagonal  pigmented 
epitheUum  will  have  come  away  with  the  rest  of  the  retina,  and  in 
consequence  of  the  rupture  of  some  of  the  cells  the  preparation  will 
be  strewn  with  pigment  granules  which,  like  all  minute  granules 
suspended  in  fluid,  exhibit  very  strikingly  the  Brownian  moleciilar 
movement.  Some  of  the  pigment  cells  may  be  observed  intact, 
either  isolated  or  in  patches.  If  seen  in  profile,  it  may  be  noticed 
that  near  one  surface  (the  oiiter)  the  cell  is  almost  entirely  free 
from  black  pigment,  while  fi^om  the  other  fine  streamers  of  the 
cell-protoplasm,  dotted  with  pigment  granules,  extend.  In  their 
natural  position  these  pass  between  and  amongst  the  outer  segments 
of  the  rods  in  the  eyes  of  an  animal  which  has  been  kept  in  the 
light,  biat  are  confined  to  the  region  of  the  cell-nuclei  in  eyes  which 
have  been  kept  in  the  dark,  or  which  are  taken  from  a  curarised 
annual   (Kiihne).     The   cones   also   vary   in    length   under    these 


278  PRACTICAL  HISTOLOGY 

conditions,  being  greatly  shortened  in  the  eye  of  an  animal  kept  in 
the  light  (Engelmann).  This  difference  is  best  shown  in  sections 
of  retinae  which  have  been  hardened  in  corrosive  sublimate. 

The  retina  of  a  bird,  of  a  reptile  (tortoise),  and  of  a  fish  may  be 
teased  out  fresh  in  vitreous  humour,  in  the  same  way  as  that  of 
the  frog.  The  chief  points  of  interest  in  these  preparations  are  the 
ellipsoid  or  lenticular  bodies  in  the  inner  segments  of  the  rods 
(bird  and  amphibian)  and  cones  (bird,  reptile,  and  amphibian),  the 
bright,  fatty  globules  of  different  colours  in  the  inner  segments  of 
the  cones  in  the  tortoise  and  bird,  and  the  twin  or  double  cones, 
especially  large  in  the  fish's  retina.     The  various  other  points  in 

which  the  retina  in  these  animals  differs  from  that  of  mammals, 
may  be  studied  by  employing  the  same  methods  of  preparation  as 

or  the  mammalian  retina. 

THE   LENS   AND   VITREOUS   HUMOUR 

The  lens  fibres. — The  following  will  be  found  the  best 
mode  of  isolating  the  fibres  of  the  lens,  as  well  as  for  showing 
their  arrangement.  Take  the  fresh  eye  of  any  animal — that 
of  the  ox  or  sheep  for  example — and  cut  it  across  into  an 
anterior  and  a  posterior  half.  Place  the  anterior  part,  having 
removed  what  remains  of  the  vitreous  humour,  in  one-third 
alcohol.  Then  scratch  through  the  posterior  capsule,  which  is 
readily  ruptured  and  curls  away  from  the  lens  proper.  This 
can  easily  be  shelled  out,  and  is  left  in  the  fluid,  the  remainder 
of  the  eye  being  rejected.  The  lens  is  allowed  to  remain  in 
the  alcohol  for  two  or  three  days,  being  merely  turned  over 
once  or  twice.  It  will  be  found  that  its  substance  tends  both 
to  separate  along  the  radiating  lines  which  mark  the  planes 
of  junction  of  the  ends  of  the  fibres,  and  also  to  peel  into  con- 
centric lamellse  like  the  coats  of  an  onion  ;  and,  if  a  piece  of 
one  of  these  lamellse  is  taken  up  with  the  forceps,  it  will  tear 
in  the  direction  of  the  fibres  from  one  of  the  planes  of  junc- 
tion of  the  anterior  surface  to  the  corresponding  plane  of  the 
posterior.  The  fibres  can  be  readily  separated  with  needles. 
For  this  purpose  portions  should  be  taken  both  from  the 
superficial  and  from  the  more  central  parts  of  the  lens.     In 


THE  EYE  279 

many  of  the  superficial  fibres  a  round  or  elongated  nucleus 
may  be  detected  at  one  part,  and  since  the  nuclei  of  adjacent 
fibres  are  met  with  in  about  the  same  region,  when  a  number 
of  fibres  are  seen  together  the  nuclei  lie  in  an  irregular  row. 
The  riband  like  shape  of  the  fibres  may  be  made  out  at  parts 
where  they  are  turned  over  so  as  to  be  seen  edgeways. 

Sections  of  the  lens. — For  cutting  sections  of  the  lens  it  is 
best  to  harden  it  in  formaldehyde  (5  to  10  per  cent,  formol). 
The  whole  anterior  half  of  an  eye  should  be  put  in  this,  the 
cornea  having  been  partly  removed  so  as  to  enable  the  fluid 
to  get  freely  to  the  front  as  well  as  to  the  back  of  the  lens, 
but  the  capsule  is  not  to  be  ruptured.  After  two  or  three 
days,  the  preparation  may  be  soaked  in  gum,  and  sections  may 
be  made  by  the  freezing  process.  They  are  to  be  mounted 
in  glycerine.  The  lens  must  not  be  put  in  spirit  to  complete 
the  hardening,  for  strong  spirit  renders  the  tissue,  especially 
the  central  parts,  quite  hard  and  horny  in  consistence,  and 
the  outlines  of  the  fibres  become  obliterated. 

The  epithelium  of  the  lens-capsule. — This  may  have  been 
seen  in  the  antero-posterior  section  as  a  single  row  of 
nucleated  cells,  lying  immediately  behind  the  anterior  part  of 
the  capsule.  To  show  it  on  the  flat  it  is  to  be  stained  with 
nitrate  of  silver.  With  this  object  a  lens  still  enclosed  in  its 
capsule  is  removed  from  a  fresh  eye,  and,  after  having  been 
rinsed  in  distilled  water,  transferred  for  five  minutes  to  h  per 
cent,  nitrate  of  silver  solution.  It  is  then  again  washed  with 
distilled  water,  and  placed  in  the  light  in  weak  spirit  (equal 
parts  spirit  and  water).  When  brown  it  is  removed  from  the 
light,  and  placed  in  75  per  cent,  spirit.  After  twenty-four 
hours  it  is  hard  enough  to  allow  tangential  sections  to  be 
made  from  the  anterior  surface,  to  include  the  capsule,  the 
epithelium,  and  the  parts  of  the  lens  substance  immediately 
subjacent  to  this.  The  sections  are  mounted  either  in  glycerine 
or  in  balsam  with  the  brown  surface  uppermost  :  and  through 
the  elastic  capsule,  which  is  not  distinctively  stained,  the  outlines 
of  the  epithelium-cells  are  clearly  seen.     At  places  the  silver 


280  PEACTICAL  HISTOLOGY 

solution  may  have  penetrated  to  the  superficial  lens  fibres,  and 
will  be  found  to  have  stained  the  cementing  substance  between 
them. 

The  zonule  of  Zinn  and  the  hyaloid  membrane  of  the 
vitreous  humour. — Take  the  anterior  half  of  the  eye  (preserved 
in  spirit  or  formol)  of  an  albino  rabbit,  and  having  pinned  it, 
the  cornea  downwards,  on  a  loaded  cork,  and  removed  the 
remains  of  the  vitreous  humour,  gently  seize  the  lens  with  fine 
forceps,  and  draw  it  away  from  the  iris.  In  doing  this  it  will 
drag  with  it  the  suspensory  ligament,  the  zonule  of  Zinn,  and 
the  part  of  the  hyaloid  membrane  continuous  with  this,  so 
that  the  separated  lens  appears  girdled  by  a  delicate,  some- 
what crumpled-looking,  membranous  zone,  closely  adherent 
at  its  inner  border  to  the  equator  of  the  lens,  and  bounded 
outwardly  by  a  ragged  margin — the  torn  edge  of  the  hyaloid. 
Cut  out  with  fine  scissors  a  segment  of  this  zone,  including 
its  whole  breadth,  and  with  a  section-lifter  transfer  the  piece 
so  removed  to  hsemalum  solution  or  some  other  dye.  When 
sufficiently  stained — and  it  stains  very  readily — transfer  it  to 
a  dish  of  water,  and  from  this  float  it  on  to  a  slide,  avoiding 
all  creases  except  of  course  the  natural  ones  of  the  zonule. 
It  may  then  be  covered,  and  the  water  in  which  it  is  mounted 
replaced  by  glycerine.  Or,  instead  of  placing  it  in  the  water, 
it  may  be  transferred  from  the  logwood  to  spirit,  and  then 
passed  through  oil  of  cloves  and  mounted  in  balsam.  These 
preparations  exhibit  well  the  folds  and  striations  of  the  zonule, 
and  the  rounded  corpuscles,  like  white  blood -corpuscles,  which 
are  dotted  here  and  there  over  the  surface  of  the  hyaloid. 

THE    BLOOD-VESSELS    OF    THE    EYE 

For  the  demonstration  of  the  blood-vessels  the  head  of  an 
albino  rabbit  should  be  injected,  a  cannula  being  placed  in 
each  carotid,  and  the  two  cannulas  connected  to  the  arms  of 
a  Y-shaped  tube,  the  stem  of  which  is  brought  into  communi- 
cation  by   an   indiarubber   tube   with    the   injection   bottle. 


BLOOD-VESSELS   OF  THE   EYE  281 

After  the  blood  has  been  driven  out  of  the  vessels,  before  the 
flow  of  injection  fluid,  the  neck  of  the  animal,  just  below  the 
place  where  the  cannulas  are  inserted,  is  surrounded  by  a  loop 
of  wire,  which  is  drawn  as  tightly  as  possible  to  pre\ent  the 
escape  of  the  injection  ;  and  the  pressure  is  then  raised  to 
about  four  inclies  of  mercury  and  kept  there  for  some  minutes, 
so  as  to  make  certain  that  all  the  blood-vessels  shall  be 
completely  filled.  The  whole  is  then  allowed  to  stand  and 
become  cold,  that  the  gelatine  may  set,  after  which  the  eyes 
are  to  be  carefully  excised,  and  placed  in  3  p.c.  bichromate,  free 
incisions  being  made  in  the  sclerotic.  After  two  or  three 
weeks  they  are  tranferred  to  spirit,  first  50  per  cent.,  then 
75  per  cent.,  then  strong.  When  they  have  been  in  this  last 
a  day  or  two  the  following  parts  may  be  prepared  : — 

The  conjunctival  vessels,  and  the  subconjunctival  vessels 
of  the  sclerotic. — By  making  with  a  razor  held  in  the  hand 
and  wetted  with  spirit  a  tangential  section  from  the  region  of 
the  corneo-sclerotic  junction,  and  after  passing  the  piece  s<j 
obtained  through  oil  of  cloves,  mounting  it  in  balsam  with 
the  outer  surface  uppermost,  the  distribution  of  the  vessels 
is  exhibited,  both  in  the  conjunctiva  and,  by  focussing  more 
deeply,  those  in  the  sclerotic  at  and  near  the  margin  of 
the  cornea.  Another  plan  consists  in  cutting  away  a  small 
piece,  including  the  whole  thickness  of  both  cornea  and 
sclerotic,  and  mounting  in  a  similar  way.  The  thickness  and 
irregxilarity  of  the  piece  so  obtained  is  a  disadvantage,  but,  on 
the  other  hand,  the  canal  of  Schlemm  and  the  other  venous 
sinuses  may  be  observed,  if  the  injection  has  been  a  successful 
one,  by  focussing  still  lower  than  for  the  looped  vessels  of  the 
sclerotic. 

Vessels  of  the  choroid  and  iris.— One  of  the  two  injected 
eyes  is  to  be  divided  by  an  antero-posterior  cut  with  the  razor 
into  a  right  and  a  left  half.  One  of  the  two  halves,  the  one 
which  does  not  include  the  attachment  of  the  optic  nerve,  is 
first  taken,  and  the  vitreous,  retina,  and  lens  removed,  so  as 
to  clear  the  inner  sui'face  of  the  choroid  and  iris.     The  last- 


282  PEACTICAL  HISTOLOaY 

named  are  next  to  be  separated  as  one  piece  of  membrane 
from  the  sclerotic.  The  piece  so  obtained  is  then  to  be  again 
divided  into  two,  by  another  antero-posterior  cut  with  the 
scissors,  and  the  resulting  halves  are  to  be  mounted,  after 
passing  as  usual  through  oil  of  cloves,  in  balsam,  the  one  with 
the  inner  and  the  other  with  the  outer  surface  uppermost. 
Each  includes,  of  course,  the  fourth  part  of  the  choroid  coat 
with  some  of  the  ciliary  processes,  and  a  piece  of  the  iris  ;  and 
with  a  low  power  the  course  and  disposition  of  the  blood- 
vessels in  these  parts  can  be  readily  followed.  Besides  these 
comprehensive  preparations,  separate  ones  may  be  made  from 
the  other  half  of  the  eye  of  a  portion  of  the  iris  (this  is 
rendered  more  instructive  by  lightly  staining  it),  and  one  or 
two  of  the  ciliary  processes  snipped  off  with  sharp  scissors, 
and  mounted  so  as  to  be  seen  in  profile. 

The  vessels  of  the  retina. — If  the  other  injected  eye  be 
cut  into  an  anterior  and  a  posterior  half,  and  the  posterior 
part  is  examined  after  removal  of  the  vitreous  humour,  the 
blood-vessels  will  be  seen  spreading  out  from  the  centre  of  the 
colliculus  of  the  optic  nerve.  To  exhibit  their  finer  distribu- 
tion in  the  retina,  a  piece  is  mounted  flat  in  balsam  with- 
out previous  staining,  while,  to  show  the  extent  of  their  dis- 
tribution in  the  retinal  layers,  vertical  sections,  which  need 
not  be  very  thin,  may  be  made  from  a  piece  embedded  in 
paraffin  in  the  ordinary  way,  and  similarly  mounted,  without 
staining,  in  balsam. 


283 


CHAPTER  XXII 

THE    EAR 

The  only  parts  of  the  ear  which  require  special  directions 
for  their  preparation  are  the  semicircular  canals  and  the 
cochlea. 

The  semicircular  canals. — To  study  the  structure  of  the 
membranous  semicircular  canals,  those  of  the  cartilaginous 
fishes,  e.g.  the  skate,  are  chosen.  The  skull,  which  can  be 
readily  cut  with  a  scalpel  or  strong  pair  of  scissors,  is  opened 
quite  anteriorly,  where  it  is  occupied  merely  by  a  quantity  of 
cerebro-spinal  fluid,  and  the  opening  is  extended  backwards  by 
removing  the  roof  bit  by  bit,  until  the  whole  of  the  upper 
surface  of  the  brain  is  exposed.  Two  thick  cartilaginous 
masses  will  be  seen,  one  on  either  side,  near  the  posterior 
part ;  the  large  auditory  nerves  pass  through  a  foramen  in 
each  into  their  interior.  These  masses  enclose  the  mem- 
branous labyrinth,  consisting  in  these  animals  of  utricle, 
saccule,  and  semicircular  canals,  all  of  large  size,  and  contained 
in  corresponding  cavities  and  canals,  in  the  substance  of  the 
cartilage,  but  of  which  no  trace  can  at  present  be  made  out. 
If,  however,  horizontal  slices  are  made  with  a  scalpel,  one  of 
the  canals  will  soon  be  exposed,  and  this  can  then  be  followed 
in  both  directions  by  cutting  the  cartilage  away  so  as  to  expose 
the  included  membranous  canal  in  its  whole  length.  It  will 
be  found  to  lead  at  either  end  into  a  large  membranous  bag — 
the  utricle — with  which  the  two  other  canals  also  communicate 
and  from  which  they  can  be  traced  in  the  same  manner. 
Besides  the  utricle,  there  is  another  smaller  membranous  bag 


284  PEACTICAL  HISTOLOGY 

— the  saccule — and  both  contain  a  white,  pasty,  cretaceous, 
otolithic  mass,  which  lies  over  the  part  to  which  the  nerve 
proceeds.  Near  one  of  the  attachments  of  each  semicircular 
canal  to  the  utricle  is  its  dilated  part,  or  ampulla,  and  a 
branch  of  the  auditory  nerve  may  be  seen  proceeding  to  each 
of  these,  and  terminating  abruptly  in  a  forked  thickening, 
which  indents  the  membranous  wall  and  lies  transversely  to 
the  axis  of  the  ampulla. 

The  three  ampullae,  and  the  adjacent  portions  of  the  semi- 
circular canals,  are  now  to  be  removed  from  the  cavities  con- 
taining them,  and  are  to  be  placed,  one  in  Flemming's  or 
Hermann's  solution,  one  in  corrosive  sublimate  or  corrosive 
sublimate  and  picric  acid  (p.  18),  and  the  third  in  1  per  cent, 
osmic  acid.  The  one  in  Flemming's  or  Hermann's  solution  is 
transferred  to  weak  spirit  after  three  days,  and  in  twenty-four 
hours  more  to  strong  spirit.  After  another  day  or  two  it  may 
be  stained  with  Kleinenberg's  logwood  or  some  other  bulk  stain, 
and  then  imbedded  in  paraffin.  Sections  are  to  be  made  both 
of  the  semicircular  canal  proper  and  of  the  ampulla,  oppo- 
site to  and  including  the  entrance  of  the  nerve.  The  one  in 
corrosive  sublimate  is  transferred  for  a  few  hours  first  to 
iodised  spirit,  and  then  to  absolute  alcohol ;  after  a  day  or  two 
in  this  it  may  be  embedded  and  cut.  The  sections  can  be 
stained  after  being  fixed  with  water  to  a  slide.  The  third  am- 
pulla, which  was  placed  in  osmic  acid,  is  transferred  in  an  hour 
or  two  to  water,  and  after  another  hour  to  a  mixture  of  equal 
parts  of  glycerine,  alcohol,  and  water.  After  a  day  or  two  in 
this  it  is  broken  up  in  dilute  glycerine,  and  examined  with  a 
high  power,  with  the  view  of  observing  the  two  kinds  of 
epithelium  cells — columnar  and  spindle-shaped^which  occur, 
and  the  stiff,  hair-like  projections  which  are  attached  to  the 
former.  The  ampullee  may  also  be  prepared  by  the  Golgi  method 
to  show  the  nerve-terminations. 

Sections  of  the  cochlea. — On  account  of  the  thinness  of  its 
osseous  parietes,  the  ease  with  which  it  may  be  obtained 
separate  from  the  surrounding  bone,  and  its   comparatively 


THE  COCHLEA  286 

large  number  of  spiral  turns,  the  cochlea  of  the  guinea-pig 
offers  far  greater  facilities  for  study,  and  especially  for  the 
preparation  of  sections,  than  that  of  any  other  readily  available 
animal.  The  following  is  the  mode  of  finding  and  procuring 
it  : — In  the  recently  killed  animal  the  aperture  of  the  mouth 
is  prolonged  backwards  on  either  side,  by  cutting  through  the 
cheeks  and  temporal  muscles  with  strong  scissors.  The  lower 
jaw  is  then  seized  and  forcibly  torn  away  from  the  rest  of  the 
head,  so  that  the  base  of  the  skull  is  exposed.  Here  will  be 
seen  on  either  side,  just  behind  the  fossa  for  the  articulation 
of  the  condyle  of  the  jaw,  a  large  white  bony  projection  — 
the  tympanic  bulla.  This  is  not  yet  to  be  opened,  but  the 
cartilaginous  external  auditory  meatus  is  first  cut  through, 
and  with  the  aid  of  bone-forceps  or  strong  scissors,  the  bulla 
in  question,  together  with  the  petrous  bone  to  which  it  is 
attached,  is  separated  from  the  rest  of  the  skull.  In  a  young 
animal  this  can  be  very  readily  effected,  simply  by  inserting  a 
strong  blunt  instrument  into  the  base  of  the  skull  just  in  front 
of  the  bulla,  and,  using  it  as  a  lever,  raising  the  bone  and 
forcing  it  away  from  its  attachments.  The  bones  of  either  side 
being  thus  removed,  the  adhering  soft  parts  are  cleared  away 
and  the  bulla  is  broken  open  at  its  most  prominent  part.  On 
now  looking  into  the  cavity  there  will  be  noticed  on  one  side 
the  delicate  tympanic  membrane  stretching  over  the  end  of  the 
external  meatus,  with  the  handle  of  the  malleus  attached  to  it, 
and  on  the  opposite  wall  a  well-marked  conical  projection, 
which  there  is  no  difficulty  in  recognising  as  the  cochlea  ; 
indeed,  its  bony  wall  is  so  thin  that  it  is  possible  to  count  the 
number  of  turns  (four)  which  it  presents.  By  cutting  the 
bulla  round  with  strong  scissors,  the  two  parts — one  in- 
cluding the  tympanic  membrane,  and  the  other  the  cochlea — 
are  separated  from  one  another,  and  the  membrane  part 
may  at  once  be  dropped  into  5  p.c.  formol  and  put  aside  to  be 
subsequently  stained  and  mounted.  From  the  other  part  as 
much  as  possible  of  the  substance  of  the  petrous  bone  is 
snipped  away,  bit  by  bit,  from  around  the  base  of  the  cochlea 


286  PEACTICAL  HISTOLOGY 

-with  scissors  or  bone  forceps,  but  great  care  should  be  taken 
in  approaching  the  cochlea  itself,  as  this  is  very  readily  split. 
When  the  surrounding  bone  has  been  in  this  way  removed, 
the  cochleas  are  dropped  into  Flemming's  or  Hermann's  fluid 
or  corrosive  sublimate  solution  to  which  is  added  an  excess  of 
crystals  of  picric  acid.  When  the  bone  is  completely  softened, 
a  process  which  is  much  facilitated  by  frequent  disturbance 
of  the  fluid,  the  cochleas  are  placed  in  50  per  cent,  alcohol 
and  transferred  gradually  to  stronger  spirit,  which  is  repeatedly 
changed.  When  the  excess  of  picric  acid  is  entirely  removed 
they  may  be  embedded  and  cut  in  a  plane  running  through 
the  axis  of  the  modiolus.  The  cochlea  may  have  been  stained 
in  bulk  before  being  embedded,  or  the  individual  sections  may 
be  stained  after  being  fixed  on  the  slide.  Only  a  few  complete 
axial  sections  can  be  obtained  from  each  cochlea. 

Teased  preparations  of  the  cochlea. — Successful  sections 
will  show  the  general  position  and  relations  of  the  rods  and 
other  parts,  and  to  a  certain  extent  the  individual  elements. 
But  only  a  profile  view  can  in  this  way  be  obtained,  and  since 
the  minute  structure  of  the  elements  composing  the  organs  of 
Corti  can  only  be  properly  seen  when  isolated,  it  is  necessary 
to  prepare  other  cochleas  with  this  object  in  view.  Another 
animal  is  accordingly  sacrificed,  and  the  cochleas  removed  as 
before.  One  is  placed  in  a  1  or  2  per  cent,  solution  of  osmic 
acid  :  the  other  in  a  0'05  per  cent,  solution  of  chromic  acid  ; 
but,  before  dropping  them  into  their  respective  fluids,  the  bony 
wall  must  be  cut  through  here  and  there  with  a  scalpel,  so 
that  the  fluid  shall  at  once  penetrate  to  the  interior  of  the 
turns.  The  cochlea  in  osmic  acid  is  removed  after  three  or 
four  hours  and  placed  in  water.  After  two  or  three  days  both 
cochleas  may  be  further  prepared  in  the  following  way  : — ■ 

The  uppermost  turn  is  broken  or  snipped  ofi"  with  scissors, 
placed  in  a  drop  of  water  on  a  slide,  the  shell  of  bone  which 
forms  the  cupola  and  outer  wall  removed,  and  the  piece  of 
lamina  spiralis  examined  with  a  low  power  (without  covering 
the  preparation)  in  order  to  learn  to  recognise  the  structures 


THE   COCHLEA  287 

which  lie  on  it.  The  glass  slide  is  then  removed  to  the  dis- 
secting microscope,  and  with  very  fine  needles  the  lamina 
spiralis  is  separated  from  the  columella,  which  is  then  rejected. 
Next  all  the  parts  on  the  lamina,  but  especially  the  row  of 
rods  of  Corti,  to  which  the  hair-cells  as  a  rule  cling,  are 
broken  up  finely,  but  at  the  same  time  slowly  and  carefully, 
the  preparation  being  examined  now  and  again  with  the  high- 
est power  which  it  is  safe  to  use  without  a  cover-glass.  One 
of  the  chief  difficulties  is  apt  to  arise  from  portions  of  the 
tissue  sticking  to  the  needles  :  if  this  is  the  case,  pieces  of 
glass  rod  drawn  out  to  a  tine  point  may  be  substituted. 
When  the  more  important  parts  have  been  broken  up  pretty 
completely,  any  thick  pieces  of  tissue  unimportant  to  the 
present  observation,  such  as  bits  of  bone,  or  periosteum, 
bundles  of  medullated  nerve  fibres,  &c.,  should  be  picked  out, 
and  then  a  cover-glass  laid  on  and  the  preparation  examined. 
To  preserve  either  preparation  permanently  glycerine  may  be 
allowed  to  diffuse  in  at  the  edge  of  the  cover-glass  ;  but  the 
chromic  specimen  should  first  be  treated  with  a  drop  of  picro- 
carmine  solution,  so  that  the  elements  are  somewhat  stained. 

In  this  way  a  number  of  specimens  may  be  obtained  from 
each  cochlea — proceeding  from  above,  down,  and  preparing 
turn  after  turn  ;  and  careful  sketches  should  be  made  of  the 
different  structures  met  with,  and  their  arrangement  with 
regard  to  one  another.  It  will  be  found  that  the  osmic  pre- 
parations serve  best  for  showing  the  lamina  reticularis  and 
the  lamina  basilaris,  and  the  chromic  preparations  for  the 
hair-cells  and  the  niembrana  tectoria  ;  the  other  structures 
are  almost  equally  well  seen  in  both  kinds  of  preparations. 
The  large  fat-droplets  in  some  of  the  epithelium  cells  of  the 
uppermost  turn  are  peculiar  to  the  guinea-pig,  as  is  also  the 
arched  projection — seen  in  the  sections — at  the  part  where 
these  cells  are  found.  The  fat-drops  are  stained  black  in  the 
osmic  preparation. 


288  PKACTICAL  HISTOLOGY 


THE    OLFACTORY    ORGAN 

Sections  of  the  olfactory  mucous  membrane. — Very  small 
pieces  of  the  upper  turbinatebones,  or  from  the  upper  (olfactory) 
region  of  the  septum  nasi,  from  the  dog  or  rabbit,  are  placed, 
one  piece  in  a  quantity  of  0-2  per  cent,  chromic  acid  solution  or 
in  Flemming's  solution,  a  second  in  0'2  per  cent,  bichromate  of 
potash,  or  0*0.5  per  cent,  chromic  acid,  and  a  third  inl  per  cent, 
osmic  acid  for  three  or  four  hours  and  then  in  water.  The 
one  in  chromic  acid  or  Flemming  may  I'emain  a  week,  when 
it  is  transferred  to  weak  spirit  and  then  gradually  to  strong 
spirit.  After  a  day  or  two  in  this,  vertical  sections  are 
prepared  from  it. 

Isolation  of  olfactory  epithelium  cells. — ^The  other  pieces 
are  examined  after  forty-eight  hours'  maceration,  small  pieces 
being  teased  out  so  as  to  isolate  the  epithelium  cells  (both 
columnar  and  spindle-shaped),  and  if  possible,  especially  in 
the  osmic  preparations,  to  study  the  connection  of  their 
branching  lower  ends  with  subjacent  structures.  These  pre- 
parations can  be  preserved  with  glycerine,  the  chromate  ones 
being  stained  with  logwood. 

Teased  preparations  should  also  be  made  of  the  olfactory 
mucous  membrane  of  the  frog  or  newt.  Having  cut  off  the 
head  of  the  animal,  and  slit  up  the  nostrils  with  fine  scissors, 
place  the  head  in  a  quantity  of  0"2  per  cent,  solution  of 
bichromate  of  potash  or  O'Oo  per  cent,  chromic  acid.  After 
two  days,  preparations  of  the  epithelium  from  both  the 
anterior  and  posterior  part  of  the  passage  may  be  made. 
The  cells  are  obtained  with  the  greatest  ease  by  scraping 
the  mucous  surface  with  the  point  of  a  scalpel,  and  shaking 
out  the  material  in  a  drop  of  water  on  a  slide.  A  piece  of 
hair  is  added,  and  the  preparation  covered  and  examined. 
In  the  portion  obtained  from  near  the  anterior  nares  ordi- 
nary columnar  ciliated  epithelium  cells  will  be  seen.  In  that 
from  the   true   olfactory  part   the   cells,  although   many  of 


TASTE-BUDS  280 

them  !iro  of  a  columnar  form,  are  destitute  of  cilia,  ami  in 
addition  to  the  columnar  elements,  spindle-shaped  (olfactory) 
colls  are  met  with  which  are  provided  with  a  bunch  of  stiff- 
looking,  hair-like  processes,  resembling  the  similar  .ippondages 
of  the  auditory  epithelium. 

The  connection  of  the  olfactory  cells  with  the  olfactory 
nerve-iil)res  may  be  studied  in  preparations  from  fretal  animals 
made  by  the  method  of  Golgi. 

THE   GUSTATORY    ORGANS 

Taste-buds.— For  studying  the  taste-buds,  the  foliated 
papilhv  which  are  found  on  either  side  of  the  base  of  the 
rabbit's  tongue  are  used.     To  obtain  them  the  tongue  is  cut 

Fig.  59 


Tongue  of  rabbit,  seen  from  above 
;),  p.  Papilla?  foliata> 


out  entire  from  the  recently-killed  animal  ;  when  the  little 
oval  patches  marked  with  transverse  ridges  may  readily  be 
found  (fig.  59,  2h  P)-  They  are  snipped  off  with  curved 
scissors,  and  one  is  dropped  into  a  mixture  of  one  part  of  1 
per  cent,  osmic  acid  and  three  parts  of  Miiller's  fluid,  and  the 
other  is  placed  in  1  per  cent,  osmic  acid.  After  three  days  the 
one  in  osmic-bichromate  is  divided,  and  one  half  is  prepared 
with  silver  nitrate  by  Golgi's  method,  whilst  the  other  half  is 
placed  in  strong  spirit.  Both  halves  are  eventually  cut  into 
sections,  vertical  to  the  surface  of  the  mucous  membrane,  and 
across  the  direction  of  the  ridges. 


290  PEACTICAL  HISTOLOGY 

The  piece  that  was  placed  in  osmic  acid  is  transferred  to 
water  after  three  or  four  hours,  and  may  then  be  used  as 
follows  : — In  the  first  place,  two  or  three  sections  are  to  be 
obtained  like  those  made  from  the  other  piece  ;  they  can  be 
made  by  the  freezing  method,  and  are  to  be  mounted  in 
glycerine.  One  such  section  is  to  be  placed  in  a  drop  of 
water  on  a  slide,  and  an  attempt  made  with  needles,  under 
the  dissecting  microscope,  to  separate  some  of  the  taste- 
buds  from  the  surrounding  epithelium.  For  this  purpose 
the  needles  must  be  very  fine,  sharp,  and  clean,  and 
the  lens  used  as  high  as  is  consistent  with  convenience  of 
manipulation.  When  one  or  more  taste-buds  have  been  thus 
separated,  the  rest  of  the  section  is  removed,  and  the  isolated 
buds  are  broken  up  as  completely  as  possible  into  their  con- 
stituent cells.  The  specimen  may  then  be  covered,  and 
a  drop  of  glycerine  allowed  to  diffuse  in  under  the  edge  of  the 
cover ;  after  which  an  examination  of  the  preparation  may  be 
made,  at  first  with  the  ordinary  high  power,  and  afterwards 
with  an  immersion  objective. 


INDEX 


A  BSORPTION  of  fat,  226 
■H     Acetic  acid,  use  in  histology,  16 
Acid  fuchsin,  21 
Adipose  tissue,  112 

development  of,  113 

Alcohol,  use  in  histology,  17 
Alkanet    for     injecting    lymphatics, 

192 
Ammonia  bichromate,  for  hardening 

nervous  tissue,  252 
Aniline,  21 

—  blue-black,  for  staining  nervous 
tissue,  253 

—  blue  (Nicholson's  No.  1),  for  stain- 
ing parietal  cells,  191 

—  dyes,  21 

Appliances  for  microscopic  work,  51 

Areolar  tissue,  00 

action  of  acetic  acid  on,  99 

corpuscles,  97 

staining  of,  99 

constrictions  on    fibre   bundles, 

100 

elastic  fibres,  97.  10(i 

stained  with  magenta,  100 

fibres  of,  96 

interstitial  injection  of  gelatine 

into,  101 

preparation  by  method  of  loca- 
lised oedema,  100 

prepared  with  nitrate  of  silver, 

104 

Arytenoid  cartiUige  of  ox,  121 

Asphalte  solution  for  injecting  lym- 
]ihatics,  192 

Attraction  particles,  Hennegtiey's 
method  of  showing,  88 

Axis-cylinders  in  sjiinal  cord,  151 


BERLIN-BLUE,  mode  of  preparin< 
179 

reduction  of,  in  tissues,  Ibi 


Berlin-blue,  solution  for  injecting 
lymphatics,  192 

Betlie,  method  of  fixing  mcthylenc- 
blue  preparations,  160 

Bichromate  of  potash  as  a  fixing  solu- 
tion, 16 

annnonia,  10 

Bismarck-brown.     Sf:e  Vcsuvin 

Bladder.     See  Urinary  bladder 

—  of  frog,  135 

Blood,  human,  action  of  reagents  on, 
58 

acetic  acid,  60 

alkalies,  01 

cliloroform,  61 

tannic  acid,  61 

— water,  59 

mode  of  obtaining,  48 

coverglass  preparation  of,  80 

on  warm  stage,  55 

effect  of  superheating,  57 

Blood-corpuscles,  human,  red,  appear- 
ance varies  with  distance  of  objec- 
tive, 50 

effect  of  reagents,  58 

•  —  salt  upon,  51 

■  —  warmth  upon,  57 

water,  59 

observation  of,  49 

structure  of,  58 

■ method  of  fixing  and  pre- 
serving, 79 

mode  of  counting,  63,  06 

human,  white,  51 

amreboid  movementsof,  57 

development  of,  113,  182 

of  frog  or  newt,  66 

action  of  electric   shocks 

on,  71 

action  of  reagents  on,  74 

iodine,  74 

feeding  of  white  corpus- 
cles, 6i» 

u2 


292 


PEACTICAL  HISTOLOaY 


Blood  corpuscles  of  frog,  migration  of 
white  corpuscles,  70 

—  influence   of  warmth,  on 

white  corpuscles,  71 

Stirling's  method  of  pre- 
serving, 81 

of  newt,  action  of  boracic  acid, 

75 

carbonic  acid,  76 

steam  in  fixing,  80 

Blood  crystals,  77 

—  platelets,  52 

—  vessels,  development  of,  113 
injection  of,  176 

larger,    epithelial     lining     of, 

shown  by  nitrate  of  silver,  152 

•  elastic  layers  of,  164 

fenestrated  membrane  of,  104 

muscular  tissue  of,  164 

mode  of  hardening,  165 

sections  of,  165 

sub-epithelial  layer  of,  163 

smaller,  epithelial  cells  of,  166 

muscular  structure  and  nu- 
clei, 167 
Bone,  corpuscles  in  lacunae,  125 

—  develoj)ing,  129 

—  hard,  grinding  section  of,  123 
precautions  in  mounting,  124 

—  softened  in  hydrochloric  acid,  125 

chromic  acid,  125 

picric  and  other  acids,  127 

lamellae   and    Sharpey's  fibres, 

127 
Boxes  for  microscopic  specimens,  14 
Brownian  movement,  55 


CABINET  for  keeping  specimens  in, 
14 
Cajal,  modification  of  Golgi's  silver 
chromate  method,  152 

—  duplicate  process,  153 

—  method  of  fixing  methylene-blue, 
160 

Camera  lucida,   for   delineating   ob- 
jects, 41 
Canada  balsam,  solution  in  xylol,  25 
Cannulas  for  injecting,  mode  of  pre- 

jjaring,  182 
Carminate  of  ammonia,  20 
Carmine  gelatine  injection,  177 

—  solution  for  staining  tissues,  20 
Cartilage,  articular,  in  fresh  state,  114 
vertical  and  tangential  sections 

of,  116 
Cartilage,  cell  spaces  of,  117 
Cartilage-cells,  action  of  water  on,  114 


Cartilage-cells,  preservation  of,  115 

stained  by  chloride  of  gold,  118 

Cartilage,  costal,  120 

—  matrix,  cell  territories  of,  120 
stained  by  logwood,  121 

—  transition  between  hyaline  and 
jrellow,  121 

Caton's  apparatus  for  studying  circu- 
lation in  fish,  171 

Cedar-wood  oil,  for  use  with  immer- 
sion objectives,  5 

Celloidin.     See  Collodion 

Cell  spaces  of  connective  tissue,  104 

Central  nervous  system,  modes  of 
preparing,  252-256 

Central  tendon  of  diaphragm  x^i'e- 
pared  with  nitrate  of  silver,  189 

Centrosomes.  See  Attraction-par- 
ticles 

Cerebellum.  See  Central  nervous 
system 

Cerebrum.  See  Central  nervous  sys- 
tem 

Chloral  hydrate  for  preparing  retina, 
275 

Chloroform  for  dissolving  paraffin,  01 

Choroid  coat  of  eye,  271 

blood-vessels  of,  281 

lamina      sui^rachoroidea, 

270 

layers  of,  271 

Chromic  acid  as  a  fixing  solution,  15 

dissociant,  16 

Ciliary  motion,  action  of  reagents  on, 
91 

carbonic  acid  on,  92 

chloroform  on,  93 

warmth  on,  91 

weak  alkalies,  92 

Ciliary  muscle,  270 

Circulation  in  omentum  of  guinea- 
pig,  172 

frog's  web,  169 

lung  of  toad,  173 

mesentery  of  toad,  171 

tails    of   tadpoles    and    fishes, 

170 

tongue  of  toad,  174 

Clearing  fluids,  24 

Cochlea,  mode  of  procuring,  284 

—  precautions  for  embedding  and 
cutting,  285 

—  softening  of  osseous  parietes,  285 

—  teased  preparations  of,  285 
Cohnheim's  gold  method,  118 
Collodion  method  of  preparing   sec- 
tions, 29 

fixing  sections,  37 


INDEX 


293 


Condenser,  biiU'.s-oj'e,  use  of,  3 
used  as  diasGctin^  lens,  7 

—  snbstiigc,  8 

Conjunctiva,  blood-vessels  of,  281 
Connective  tissue  corpuscles  iu  areo- 
lar tissue,  07 

tongue  of  toad,  175 

cell-spaces  of,  101 

Cornea,  cell-spaces  of,  2(17 
mode  of  injecting,  2G8 

—  epithelium  of,  2G1 

—  mode  of  hardening,  2G0 

—  precautions  to  avoid  curling  up  of 
sections,  2C1 

—  substantia  propria  of,  2ti2 

—  of  frog,  coii)Uscles  and  nerves  of, 
203 

rabbit,  corpuscles  and  nerves  of, 

265 

isolation  of  corpuscles,  265 

nerves  of,  266 

Corneal  tulies  of  Bowman,  269 
Corneo-sclerotic  junction,  209 
Corrosive  sublimate,  as  a  fixative,  18 
Cover-glass  holder,  9 
Cover-glasses,  mode  of  averting  pres- 
sure of,  68 

—  mode  of  cleaning,  8 

fixing,  26 

by  paraflin,  25 

—  selection  of,  8 

—  forceps  for,  9 

—  jireparations,  mode  of  making,  80 

DAMMAR  varnish,  20 
Decalcification  methods,  127 
Decolourisation,  22 
Delineation    of   microscopic    objects, 

10 
Demidesiccation  method,  'JO 
Dentinal  sheaths,  211 
Dextrine   used   in   freeijing    method, 

28 
Diaphragm  of  microscope,  use  of,  8 
— for  observing  fresh  tissue's, 

85 

—  central  tendon  of,  189 

—  lymphatics  of,  1811,  190 
Directions  for  work,  l!9 
Dyes,  18 


EAR,  283 
Egg-white  for  fixing  sections  to 
slide,  37 
Ehrlich,  methvlene-blue   method  of, 

100 
applied  to  retina,  271 


Ehrlich-Biondi  triple  sluin,  2:t 
Elastic   HbroB  in  areolar   tissue,  07, 

106 
ti'ansvorsc  section  of,  107 

—  networks    of    scrouu    membrane, 
107 

artery,  lG-1 

—  tissue,  107 

Electricity,  mode  of  api)lyiiig,  71 
Embedding  in  celloidin,  29 
parafKn,  31 

—  by  gum  metliod,  202 
Endocardium,  200 

—  fibres  of  Purkinje  in,  20() 
End-plates  of  mammals,  157 
lizard,  158 

Eosin,  as  a  stain,  21 

combined  with  hmmatein,  22 

methyl-blue,  23 

toluidin  blue,  23 

Epidermis,  cells  of  horny  layer  shown 

by  potash,  81 
Epithelioid- cells,  101 

covering  tendon,  112 

Epithelium,     ciliated,     from     frog's 

mouth,  89 

gills  of  mussel,  00 

study  of  separated  cells,  94 

—  columnar,  84 

—  olfactory,  288 

—  scaly,  of  mouth,  82 

deeper  layers  of,  83 

Epithelium-cells,  fibres  in,  88 
Erectile   tissue,  mode  of  hardening, 

247 
Eye,  blood-vessels  of,  280 

—  general  mode  of  preparing,  257 

—  of  pig  as  substitute  for  human  cj  e, 
258 

Eyelids,  sections  of,  258 
Eye-piece  of  microscope,  1 


1?AT  ABSORPTION,  226 
i      Fat-cells,  112 

membrane  of,  113 

development  of,  113 

Fibrin  in  blood,  52 

Fibro-cartilage,  yellow,  of  epiglottis, 

122 

transition  to  hvaliiie,  121 

—  —  white,  122 

Fibrous  tissue,  107 

Field-glass,  1 

Flemming,      method      of      staining 

karyokinetic  figures,  87 
Flemming's  fluid,  10 
Forceps,  11 


294 


PKACTICAL  HISTOLOGY 


Formaldehyde,  17 
Pormol,  as  a  fixative,  17 
Freezing  method,  28 
Fuchsin.     See  Magenta 
Fuchsiii  acid,  21 

&ANGLIA,  sections  of,  152 
Ganglion  ceUs,  152 
Gas,  carbonic  acid,  mode  of  applying 
to  a  preparation,  76 

—  chamber,  63 
Gastric  glands,  222 

cells  of  isolated,  223 

Gelatine  injecting  fluid,  177 

mode  of  preserving,  185 

Gentian-violet,  21 

Glycerine  for  mounting,  24 

—  jelly  for  mounting,  24 
Glycogen,  its  presence  in  white  blood- 
corpuscles,  74 

—  its  presence  in  liver-cells,  236 
Goblet  cells,  86 

Gold  chloride,  methods  of  staining 
with,  118,  159 

Gold  size,  for  fixing  cover-glass,  25 

Golgi,  silver  chromate  method  of,  152 

applied  to  study  of  bile- 
ducts,  235 

central  nervous  sys- 
tem, 252 

retina,  274 

Granules  in  blood,  52 

.  —  Osier's  observations  upon,  52 

Gullet.     See  CEsoiahagus 

Gum,  used  in  freezing  method  of 
preparing  sections,  28 

for  embedding,  202 


H^MALUM,  19 
Haematein,  19 

—  combined  with  eosin,  22 
Hsematoxylin,  19 

—  acid,  19 

—  Delafield's,  19 

—  Kleinenberg's,  20 
HtBmin  crystals,  78 
Haemoglobin  crystals,  77 
Hairs,  201 

—  development  of,  203 
Hamilton's  method   of   staining   de- 
generated nerve-fibres,  256 

Hardy  and  Westbrook,  on   granules 

of  white-corpuscles,  80 
Haversian  canals  of  bone,  124 

—  fringes  of  synovial  membrane,  198 
Hayem's  fluid,  64 


Heart,  muscular  substance  of,  205 

—  lymphatics  of,  207 

—  blood-vessels  of,  207 
Heidenhain's  bulk-stain,  20 
Hepatic  cells,  286 

Hermann,  method  for  showing  karyo- 

kinetic  figures,  88 
Hermann's  fluid,  16 
Hoggan's  rings,  189 
Holmgren,  apparatus  of,  for  studying 

circulation  in  lung,  173 


TMMEESION   objectives,  mode  of 
J-     using,  5 

Inflammation  changes  in  mesentery 
of  toad,  172 

tongue  of  toad,  176 

Injected  parts,  mode  of  preparing,  188 
Injection  apparatus,  179 

—  of  an  entire  animal,  181 

blood-vessels,  176 

mode  of  killing  an  animal 

for,  181,  233 
lymphatics,  192-96 

—  mass,  mode  of  preparing,  177 

—  with  nitrate  of  silver,  185 
Injections  fluid  in  the  cold,  184 
Instruments  required  for  microscopic 

preparation,  7 
Intercellular   substance,    sho\vn    by 

silver  nitrate,  89 
Intestine,  large,  230 

—  small,  blood-vessels  of,'  227 
mode  of  hardening,  225 

—  —  nerves  of,  227-30 
Iodised  serum,  276 

Iris,  blood-vessels  of,  281 

—  diaphragm  of  microscope,  3 

—  muscular  tissue  of,  272 

—  sections  of,  269 

Iron,  micro-chemical  test  for,  24 

—  its  presence  in  liver-cells,  236 


KANTHACK  and  Hardy,  on  fixing 
granules  of  white  corpuscles,  80 
Karyokinesis,   method    of     staining, 
21,88 

fixing,  87 

Kidney,  blood-vessels  of,  243 

—  examination    in    fresh   condition, 
244 

—  mode  of  hardening,  242 

—  tubules,  isolation  of,  by  Ludwig's 
method,  243 

basement  membrane  of,  stained 

with  nitrate  of  silver,  244 


INDEX 


29i 


Klein,  method  of  showing  nerves  of 

conieiv,  '250 
Kleineiiberg's  hoemiitoxj-liii,  li) 
Koch's  (von),  mcthocl,  '213 


LABIA,  moile  of  preparing,  '217 
Lachrymal  '^hmd,  2r.O 
Lamina  fusca,  '200 

—  supraehoroidea,  270 
Larynx,  179 

Lens,  isolation  of  fibres,  27'S 

—  sections  of,  '27!) 

—  suspensory  ligament  of,  '280 

—  capsule,  epithelium  of,  '279 
Lilienfeld  and  Monti,  micro-chemical 

test  for  phosphorus,  '24 
Liver,  blood-vessels  of,  '232 

—  lymphatics  of,  235 

—  mode  of  hardening,  231 

injecting  bile-ducts,  234 

Logwood  solution  for  staining  tissues, 

19 

Kleinenberg's,  20 

Luwit,  gold  chloride  method  of,  159 
Ludwig's    appariitus     for     injecting 

lympiiatics,  193 
Lnng,  injected  with  parafSn,  212 

—  epithelium  of  air-cells,  210 

—  injection  of  blood-vessels,  211 

—  mode  of  hardening,  209 

—  of  toad,  circulation  in,  173 
Lymphatic  glands,  237 
Lymphatics,  injection  of,  192 

—  of  diaphragm,  natural  injection  of, 
196 

tendon,  mode  of  injecting,  193 

—  larger,  192 

—  smaller,  in  omentum  and  central 
tendon,  186-90 


MACALLUJM,   micro-chemical  test 
for  iron,  24 
Magenta,  21 

—  double  staining  by,  22 

—  solution  for  fresh  tissues,  22 
Magnifj'ing     power    of     microseoi)e, 

estimation  of,  47 
Mammary  glands,  251 
Mann's  fluid  for  hardening,  18 

staining,  23 

Marchi's  solution,  254 
MaiTow,  131 

—  red,  on  warm  stage,  132 

giant  cells  of,  133 

Mayer's  carmalum,  20 

—  hsemalum,  19 


Measurement  of  an  object  under  the 

microscope,  44 
Metliyl-blue,  21 

and  eosin,  23 

J\Iothyl-gi-een,  21 
Methyl-violet,  21 
Methylene-blue,  21 

method  of  Khrlich,  100 

Mesentery,   staincil    witli   nitrate   of 

silver,  IHH 

—  circulation  in,  171 
Micrometers,  44,  45 
Micrdpbotograpiiy,  44 
Microscope,  binocular,  use  of,  3 

—  eye-piece  of,  4 

—  for  dissection,  7 

—  magnifying  power  of,  40 

—  parts  of,  1 

—  powers  of,  4 

—  selection  of,  by  student,  0 
Microtome,  freezing,  29 

—  inclined  plane,  30 

—  tripod,  33 

—  rocking,  34 

—  Minot's,  30 

Migration  of   white  corpuscles   from 

veins,  176 
Modelling,  41 

Moist  chamber  made  with  putty,  01 
Mould  for  paraffin,  32 
Mouth,  mucous  membrane  of,  213 
Mucous  glands  of  tongue,  217 
Miiller's  fluid,  composition  of,  10 

used  for  eye,  257 

Muscle,  blood-vessels  of,  145 

—  ending  in  tendon  in  mouse's  tail, 
144 

frog,  144 

—  examined  by  polarised  light,  142 

—  transverse  section  of,  13'7 

—  of  insects,  139 

— mode  of  production  of  the 

transverse  striie,  140 

prepared  by  Rollett's  method, 

141 

studying  contrac- 
tion of,  140,  141 

—  involuntai-y,  mode  of  isolating  colls 
of,  134 

showing  nuclei,  134 

prepared  with  nitrate  of  silver, 

135 

—  voluntai-y,  action  of  acetic  acid, 
137 

demonstration    of    sarcolemma 

in,  137 

mammalian,  130,  137 

isolation  of  fibres,  1 3S 


296 


PEACTICAL  HISTOLOGY 


Muscle,  voluntary,  separation  of  into 

discs  and  muscle-columns,  138 
Myeloplaxes,  133 


NAIL,  cells  of,  separated  by  potash, 
84 

—  embedded  by  gum-method,  202 

—  sections  of,  202 
Needles,  mounted,  11 

—  spear-headed,  11 
Nerve  cells  of  ganglia,  152 

spinal  cord,  150 

Nerve  fibres,  degenerating,  150 
medullated,  146 

treated     with    osmic    acid, 

147 
stained  with  nitrate  of  silver, 

148 

non-medullated,  147 

Nerve  trunk,  perineurium  of,  shown 

by  silver  method,  148 
sections  of,  149 

—  —  structure  of,  148 
Nerves,  motor,  ending  of,  157 

—  —  methods  of  staining,  158 
Nervous  plexuses  of  intestine,  227- 

230 
Nervous   system,   central,   modes   of 

preparing,  252-256 
Neuroglia-cells,  150 
Nissl,  method  of,  254 
Nitromolybdate  of  ammonia  for  fixing 

methylene-blue,  21,  160 
Nose-piece,  5 
Nuijl,  fibres  in  epithelium  cells,  88 


OBJECT-GLASS,  or  objective,  4 
Objectives,  immersion,  5 

—  apochromatic,  5 
Ocular  of  microscope,  4 
CEsophagus,  220 

^  blood-vessels  of,  220 

Oil  of  bergamot,  for  clearing,  25 

turx3entine,  25 

cloves,  for    clarifying  sections, 

25 
Olfactory  mucous  membrane,  288 
Oliver,    G.,   method    for    estimating 

number  of  blood-corpuscles,  66 
Omentum,  prepared  with  nitrate  of 

silver,  186 

—  circulation  in,  172 
Optical  section,  169 
Orange  G,  21 
Organs  of  taste,  289 
Osier  on  blood-platelets,  52 


Osmic  acid,  as  a  fixing  solution,  16 

—  —  colours  fatty  substances  black, 
10 

for  nerve,  147 

for  retina,  273-275 

Ossification,  intracartilaginous,  129 

—  intramembranous,  129 

—  of  lower  jaw,  129 
Ovary,  248  ' 

Ovum,  mode  of  obtaining,  248 


PACINI'S  fluid  for  blood-corpuscles, 
79 

Pacinian  corpuscles  from  cat's  mesen- 
tery, 154 

sections  of,  157 

treatment   with   chromic   acid, 

156 

liitrate  of  silver,  156 

osmic  acid,  155 

Pal's  method.     See  Weigert-Pal 

Palate,  218 

Pancreas,  236 

Paraffin,  for  fixing  coverglass,  24 

—  method  of  jpreparing  sections,  31 

—  mould  for,  32 

—  for  embedding,  31 
Pericardium,  205 
Perineurium,  148 

Phloroglucin    in    decalcifying-    bone, 

127 
Phosphorus,  micro-chemical  test  fcr, 

24 
Pia  mater,  vessels  of,  168 
Picric  acid,  as  a  fixative,  17 
Picrocarmine,  20 

Pigment,  hexagonal,  of  retina,  276 
■ —  of    ciliary    processes,   method    of 

bleaching,  270 
Pipettes,  mode  of  making,  11 

—  measuring,  for  blood  enumeration, 
67 

Pituitary  body,  237 

Pleura,  208 

Polarisation  apparatus,  5 

Polarising  microscope,  142 

Prostate  gland,  247 

Pulmonary  vessels,  injection  of,  211 

Purkinje,  fibres  of,  206 


RANVIEE    on     Sharpey's     fibres, 
128 
Ranvier's  demidesiccation  method,  96, 

98 
—  methods    of      preparing     areolar 
tissue,  102 


INDEX 


297 


Ranvier'a  metliocl  of  whowing  teiulon- 
cells,  110 

membrane  of  fat-uell,  IIU 

Reagents  in  common  use,  14 
Rticklingliausen's  silver  method,  103 
Bemak,  fibres  of,  147 
Rcticuliir  tissue,  238 
Retina,  blood-vessels  of,  '282 

—  fibres  of  M  filler,  275,  27(i 

—  fresh  in  vitreous  humour,  276 

—  in  iodised  serum,  270 

—  isolation  of  elements,  275 

—  methods  of  hardening  and  cutting 
sections,  273-275 

—  of  bird,  reptile,  and  fish,  278 

—  of  frog,  277 

RoUett,  gold  method  of,  applied   to 

muscle,  141 
Rubin,  S.     See  Acid  fuchsin 


q AFRANIN,  21 

0     Salamander  tadpoles  for  kaiTO- 

kinetic  figures,  87 
Salivarv  eoi-puscles,  83 

—  glands,  218 

Salt  solution,  normal,  15 
Sanderson  and  Strieker,  on  circula- 
tion in  omentum,  173 
SchafTer,  on  decalcifying  methods,  128 
Sclerotic  coat  of  eye,  259 

blood-vessels  of,  281 

lamina  fusca,  2G0 

Scissors,  11 

Scrotum,  247 

Section-lifters,  11 

Sections,  modes  of  preparing,  2G 

keeping  in  series,  34 

—  methods  of  fixing  upon  slide,  3(5 
mounting,  39 

—  optical,  1(59 

Semicircular  canals,  mode  of  finding 

and  preparing,  283,  284 
Seminal  vesicles,  247 
Seminiferous  tubules,  epithelioid  cells 

of,  250 

—  —  isolation  of,  250 
Serous  membranes,  180 
Sharpey,  fibres  of,  127 
Shellac  for  fixing  sections,  87 
Sherrington,  fluid  for  diluting  blood 

for  enumeration,  64 
Silver  chromate  method.     Sec  Golgi 

—  nitrate,  method  of  staining  epithe- 
lium with,  89 

connective  tissue,  104 

Skin,  199 

—  blood-vessels  of,  201 


Skin,  double  Htuining  of,  200 

—  preparation  of,  199 

—  nerves  of,  203 

—  lymphatics  of,  203 

Slidos  for  microscopic  purposes,  h 

counting  blood-corpuscles,  (i4 

Solutions   for   examination   of   fresh 
tissues,  14 

fixing  the  tissues,  15 

staining  histological  objects,  18 

Spinal  cord,  253 

isolation  of  cells  of,  150 

Spleen,  239 

—  demonstration   of  retiforni  tissue 
of,  240 

—  injection  of,  239 
Stage  of  microscope,  1 

—  mechanical,  5 
Staining  fluids,  18 

—  double  and  triple,  22 

Steam  for  fixing  amoeboid  corpuscles, 

80 
Stirling's  method  of  staining  and  pre- 

sen'ing  frog's  blood-corpuscles,  81 
showing  medullated   nerves 

in  trachea,  212 
Stomach,  221 

—  blood-vessels  of,  228 

—  glands  of,  222,  223 

—  horizontal  sections  of,  223 

—  lymphatics  of,  224 

Stomata  in  lymphatic  septum  of  frog, 
191 

—  central  tendon  of  diaphragm,  190 
Suprarenal  capsule,  5,  241 
SynoviLil  membranes,  197 

—  —  blood-vessels  of,  198 

Haversian  fringes  of,  198 

preparation  of  by  silver  method, 

100,  197 
SjTinge,  hypodermic,  for  interstitial 

injection,  101 
for  injecting  lymphatics,  195 

—  condensing,  for  injection,  181 


fPACTlLE  corpuscles,  201 
J-      Tarsal  cartilage  of  eyelid,  2r!) 
Taste-buds  in  papiUse  foliatte  of  rab- 
bit, 289 

circumvallatas,  217 

Teeth,  in  situ,  215 

—  development  of,  216 

—  dentinal  sheaths  of,  214 

—  sections  obtained  by  grinding,  213 
— -v.  Koch's  method,  213 

—  sections  of,  softened,  214 

—  soft  tissues  of,  215 


298 


PRACTICAL   HISTOLOGY 


Tendon  of  mouse's  tail,  108 

action  of  acetic  acid  on, 

109 
cell-spaces  of.  111 

—  transverse  section,  110 

—  cells,  109 

Tenon,  capsule  of,  259 
Testis,  249 

—  lymphatics  of,  249 

—  tubules  of,  250 
Thionin,  21 

Thoma,  decalcification  method,  127 
Thymus  gland,  237 
Thyroid  body,  237 
Toluidin-blue,  21 

and  eosin,  23 

Tongue,  217 

—  blood-vessels  of,  218 

—  of  toad,  174 
Tonsils,  218 
Trachea,  212 

—  epithelium  of,  94 

Trays  for  microscopic  specimens,  14 
Tunica  vaginalis,  251 


DRETERS,  244 
—  epithelium  of,  245 
Urinary  bladder,  245 

of  frog,  135 

Uriniferous  tubules.     See  Kidney 
Uterus,  248 


YAGINA,  247 

'     Vaginal  synovial  membranes,  197 
VesiculsB  seminales,  247 
Vesuvin,  21 
Vitreous  humour,  hyaloid  membrane 

of,  280 
Villi,  structure  of,  225 


VXTALLERIAN  degeneration,  255 

''  Warming  apparatus,  with  gas 
regulator,  for  chloride  of  gold  pre- 
parations, ll9 

Warm-box,  58 

Warm  stage,  simple,  53 

mode  of  estimating  tempera- 
ture, 54 

with  gas  regulator,  55 

Weigert's  method  for  neuroglia,  255 

Weigert-Pal  method  for  central  ner- 
vous system,  253 

Weil.     See  Koch 

White  of  egg  for  fixing  sections,  37 


y  YLOL-BALSAM,  25 
-ii-    Xylol  for  clearing,  25 
dissolving  paraffin,  33,  37 


I70NULE  of  Zinn,  280 


OM551                     Schl 

1897 

Schafer 

A  course 

of  practical  histology. 

t 

m 


■:^il 


\^^■'^^ 


■:•} 


»'.n->rAnr;tJ';'?; 


